Compositions for nucleic acid delivery

ABSTRACT

A method for delivering a nucleic acid to a cell can include exposing sample cells to a composition which includes charged lipids.

CLAIM OF PRIORITY

This application claims priority to provisional U.S. Patent ApplicationNo. 61/267,419, filed Dec. 7, 2009, to provisional U.S. PatentApplication No. 61/334,398, filed May 13, 2010, and to provisional U.S.Patent Application No. 61/384,303, filed Sep. 19, 2010, each of which isincorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to lipids, lipid particles, compositionsincluding lipid particles, and methods for making and using these.

BACKGROUND

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids,immune stimulating nucleic acids, antisense, antagomir, antimir,microRNA mimic, supermir, U1 adaptor, and aptamer. These nucleic acidsact via a variety of mechanisms. In the case of siRNA or miRNA, thesenucleic acids can down-regulate intracellular levels of specificproteins through a process termed RNA interference (RNAi). Followingintroduction of siRNA or miRNA into the cell cytoplasm, thesedouble-stranded RNA constructs can bind to a protein termed RISC. Thesense strand of the siRNA or miRNA is displaced from the RISC complexproviding a template within RISC that can recognize and bind mRNA with acomplementary sequence to that of the bound siRNA or miRNA. Having boundthe complementary mRNA the RISC complex cleaves the mRNA and releasesthe cleaved strands. RNAi can provide down-regulation of specificproteins by targeting specific destruction of the corresponding mRNAthat encodes for protein synthesis.

The therapeutic applications of RNAi are extremely broad, since siRNAand miRNA constructs can be synthesized with any nucleotide sequencedirected against a target protein. To date, siRNA constructs have shownthe ability to specifically down-regulate target proteins in both invitro and in vivo models. In addition, siRNA constructs are currentlybeing evaluated in clinical studies.

However, two problems currently faced by siRNA or miRNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind RISC when administered systemically as the freesiRNA or miRNA. These double-stranded constructs can be stabilized byincorporation of chemically modified nucleotide linkers within themolecule, for example, phosphothioate groups. However, these chemicalmodifications provide only limited protection from nuclease digestionand may decrease the activity of the construct. Intracellular deliveryof siRNA or miRNA can be facilitated by use of carrier systems such aspolymers, cationic liposomes or by chemical modification of theconstruct, for example by the covalent attachment of cholesterolmolecules. However, improved delivery systems are required to increasethe potency of siRNA and miRNA molecules and reduce or eliminate therequirement for chemical modification.

Antisense oligonucleotides and ribozymes can also inhibit mRNAtranslation into protein. In the case of antisense constructs, thesesingle stranded deoxynucleic acids have a complementary sequence to thatof the target protein mRNA and can bind to the mRNA by Watson-Crick basepairing. This binding either prevents translation of the target mRNAand/or triggers RNase H degradation of the mRNA transcripts.Consequently, antisense oligonucleotides have tremendous potential forspecificity of action (i.e., down-regulation of a specificdisease-related protein). To date, these compounds have shown promise inseveral in vitro and in vivo models, including models of inflammatorydisease, cancer, and HIV (reviewed in Agrawal, Trends in Biotech.14:376-387 (1996)). Antisense can also affect cellular activity byhybridizing specifically with chromosomal DNA. Advanced human clinicalassessments of several antisense drugs are currently underway. Targetsfor these drugs include the bcl2 and apolipoprotein B genes and mRNAproducts.

Immune-stimulating nucleic acids include deoxyribonucleic acids andribonucleic acids. In the case of deoxyribonucleic acids, certainsequences or motifs have been shown to illicit immune stimulation inmammals. These sequences or motifs include the CpG motif,pyrimidine-rich sequences and palindromic sequences. It is believed thatthe CpG motif in deoxyribonucleic acids is specifically recognized by anendosomal receptor, toll-like receptor 9 (TLR-9), which then triggersboth the innate and acquired immune stimulation pathway. Certain immunestimulating ribonucleic acid sequences have also been reported. It isbelieved that these RNA sequences trigger immune activation by bindingto toll-like receptors 6 and 7 (TLR-6 and TLR-7). In addition,double-stranded RNA is also reported to be immune stimulating and isbelieve to activate via binding to TLR-3.

One well known problem with the use of therapeutic nucleic acids relatesto the stability of the phosphodiester internucleotide linkage and thesusceptibility of this linker to nucleases. The presence of exonucleasesand endonucleases in serum results in the rapid digestion of nucleicacids possessing phosphodiester linkers and, hence, therapeutic nucleicacids can have very short half-lives in the presence of serum or withincells. (Zelphati, O., et al., Antisense. Res. Dev. 3:323-338 (1993); andThierry, A. R., et al., pp 147-161 in Gene Regulation: Biology ofAntisense RNA and DNA (Eds. Erickson, R P and Izant, J G; Raven Press,NY (1992)). Therapeutic nucleic acid being currently being developed donot employ the basic phosphodiester chemistry found in natural nucleicacids, because of these and other known problems.

This problem has been partially overcome by chemical modifications thatreduce serum or intracellular degradation. Modifications have beentested at the internucleotide phosphodiester bridge (e.g., usingphosphorothioate, methylphosphonate or phosphoramidate linkages), at thenucleotide base (e.g., 5-propynyl-pyrimidines), or at the sugar (e.g.,2′-modified sugars) (Uhlmann E., et al. Antisense: ChemicalModifications. Encyclopedia of Cancer, Vol. X., pp 64-81 Academic PressInc. (1997)). Others have attempted to improve stability using 2′-5′sugar linkages (see, e.g., U.S. Pat. No. 5,532,130). Other changes havebeen attempted. However, none of these solutions have proven entirelysatisfactory, and in vivo free therapeutic nucleic acids still have onlylimited efficacy.

In addition, as noted above relating to siRNA and miRNA, problems remainwith the limited ability of therapeutic nucleic acids to cross cellularmembranes (see, Vlassov, et al., Biochim. Biophys. Acta 1197:95-1082(1994)) and in the problems associated with systemic toxicity, such ascomplement-mediated anaphylaxis, altered coagulatory properties, andcytopenia (Galbraith, et al., Antisense Nucl. Acid Drug Des. 4:201-206(1994)).

To attempt to improve efficacy, investigators have also employedlipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids. In Zelphati, O. and Szoka, F. C., J. Contr.Rel. 41:99-119 (1996), the authors refer to the use of anionic(conventional) liposomes, pH sensitive liposomes, immunoliposomes,fusogenic liposomes, and charged lipid/antisense aggregates. SimilarlysiRNA has been administered systemically in cationic liposomes, andthese nucleic acid-lipid particles have been reported to provideimproved down-regulation of target proteins in mammals includingnon-human primates (Zimmermann et al., Nature 441: 111-114 (2006)).

In spite of this progress, there remains a need in the art for improvedlipid-therapeutic nucleic acid compositions that are suitable forgeneral therapeutic use. Preferably, these compositions wouldencapsulate nucleic acids with high-efficiency, have high drug:lipidratios, protect the encapsulated nucleic acid from degradation andclearance in serum, be suitable for systemic delivery, and provideintracellular delivery of the encapsulated nucleic acid. In addition,these lipid-nucleic acid particles should be well-tolerated and providean adequate therapeutic index, such that patient treatment at aneffective dose of the nucleic acid is not associated with significanttoxicity and/or risk to the patient. The present invention provides suchcompositions, methods of making the compositions, and methods of usingthe compositions to introduce nucleic acids into cells, including forthe treatment of diseases.

SUMMARY

In one aspect, a method for delivering a nucleic acid to a cell caninclude contacting cells with a composition comprising a neutral lipidand a cationic lipid having the formula:

wherein:

R₁ and R₂ are each independently for each occurrence a C₁₀ to C₃₀ grouphaving the formula-L^(1a)-(CR^(1a)R^(1b))_(β)-[L^(1b)-(CR^(1a)R^(1b))_(β)]_(γ)-L^(1c)-R^(1c),wherein: L^(1a) is a bond, —CR^(1a)R^(1b)—, —O—, —CO—, —NR^(1d)—, —S—,or a combination thereof.

Each R^(1a) and each R^(1b), independently, is H; halo; hydroxy; cyano;C₁-C₆ alkyl optionally substituted by halo, hydroxy, or alkoxy; C₃-C₈cycloalkyl optionally substituted by halo, hydroxy, or alkoxy; —OR^(1c);—NR^(1c)R^(1d); aryl; heteroaryl; or heterocyclyl.

Each L^(1b), independently, is a bond, —(CR^(1a)R^(1b))₁₋₂—, —O—, —CO—,—NR^(1d)—, —S—, -,

or a combination thereof; or has the formula

wherein j, k, and 1 are each independently 0, 1, 2, or 3, provided thatthe sum of j, k and l is at least 1 and no greater than 8; and R^(1f)and R^(1g) are each independently R^(1b), or adjacent R^(1f) and R^(1g),taken together, are optionally a bond; or has the formula

wherein j and k are each independently 0, 1, 2, 3, or 4 provided thatthe sum of j and k is at least 1; and R^(1f) and R^(1g) are eachindependently R^(1b), or adjacent R^(1f) and R^(1g), taken together, areoptionally a bond;

or has the formula:

wherein —Ar— is a 6 to 14 membered arylene group optionally substitutedby zero to six R^(1a) groups;

or has the formula:

wherein -Het- is a 3 to 14 membered heterocyclylene or heteroarylenegroup optionally substituted by zero to six R^(1a) groups.

L^(1c) is —(CR^(1a)R^(1b))₁₋₂—, —O—, —CO—, —NR^(1d)—, —S—,

or a combination thereof.

R^(1c) is H; halo; hydroxy; cyano; C₁-C₆ alkyl optionally substituted byhalo, hydroxy, or alkoxy; C₃-C₈ cycloalkyl optionally substituted byhalo, hydroxy, or alkoxy; aryl; heteroaryl; or heterocyclyl; or R^(1c)has the formula:

R^(1d) is H; halo; hydroxy; cyano; C₁-C₆ alkyl optionally substituted byhalo, hydroxy, or alkoxy; C₃-C₈ cycloalkyl optionally substituted byhalo, hydroxy, or alkoxy; aryl; heteroaryl; or heterocyclyl.

α is 0-6; each β, independently, is 0-6; and γ is 0-6.

represents a connection between L₂ and L₁ which is:

(1) a single bond between one atom of L₂ and one atom of L₁, wherein L₁is C(R_(a)), O, S or N(O); L₂ is —(CR₅R₆)_(x)—, —C(O)—(CR₅R₅)_(x)—,—(CR₅R₆)_(x)—CR₅═CR₅—(CR₅R₆)_(y)—,—C(O)—(CR₅R₆)_(x)—CR₅═CR₅—(CR₅R₆)_(y)—, —O—, —S—, —N(Q)-, ═N—, ═C(R₅)—,—CR₅R₆—O—, —CR₅R₆—N(Q)-, —CR₅R₆—S—, —C(O)N(Q)-, —C(O)O—, —N(O)C(O)—,—OC(O)—, —C(O)—, or —X—C(R₅)(YR₃)—; wherein X and Y are each,independently, selected from the group consisting of —O—, —S—,

alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —OS(O)(Q₂)O—, and —OP(O)(Q₂)O—. R_(a) is H, alkyl, alkoxy,—OH, —N(Q)Q, or —SQ.

(2) A double bond between one atom of L₂ and one atom of L₁, wherein L₁is C; L₂ is —(CR₅R₆)_(x)—CR₅═, —C(O)—(CR₅R₆)_(x)—CR₅═, —N(Q)═, —N═,—O—N═, —N(Q)-N═, or —C(O)N(Q)-N═.

(3) A single bond between a first atom of L₂ and a first atom of L₁, anda single bond between a second atom of L₂ and the first atom of L₁,wherein L₁ is C or C(R_(a))—(CR₅R₆)_(x)—C(R_(a)); L₂ has the formula

wherein

X is the first atom of L₂, Y is the second atom of L₂, - - - --represents a single bond to the first atom of L₁, and X and Y are each,independently, selected from the group consisting of —O—, —S—,

alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —OS(O)(Q₂)O—, and —OP(O)(Q₂)O—.

Z₁ and Z₄ are each, independently, —O—, —S—, —CH₂—, —CHR⁵—, or —CR⁵R⁵—;Z₂ is CH or N; Z₃ is CH or N; or Z₂ and Z₃, taken together, are a singleC atom. A₁ and A₂ are each, independently, —O—, —S—, —CH₂—, —CHR⁵—, or—CR⁵R⁵—. Each Z is N, C(R₅), or C(R₃).

k is 0, 1, or 2; each m, independently, is 0 to 5; and each n,independently, is 0 to 5; where m and n taken together result in a 3, 4,5, 6, 7 or 8 member ring.

(4) A single bond between a first atom of L₂ and a first atom of L₁, anda single bond between the first atom of L₂ and a second atom of L₁,wherein

(A) L₁ has the formula:

wherein

X is the first atom of L₁, Y is the second atom of L₁, - - - - -represents a single bond to the first atom of L₂, and X and Y are each,independently, selected from the group consisting of —O—, —S—, alkylene,—N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O, —OC(O)O—,—OS(O)(Q₂)O—, and —OP(O)(Q₂)O—.

T₁ is CH or N; T₂ is CH or N; or T₁ and T₂ taken together are C═C; L₂ isCR₅; or

(B) L₁ has the formula:

wherein

X is the first atom of L₁, Y is the second atom of L₁, - - - --represents a single bond to the first atom of L₂, and X and Y are each,independently, selected from the group consisting of —O—, —S—, alkylene,—N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O—, —OC(O)O—,—OS(O)(Q₂)O—, and —OP(O)(Q₂)O—.

T₁ is —CR₅R₆—, —N(Q)-, —O—, or —S—; T₂ is —CR₅R₆—, —N(Q)-, —O—, or —S—;L₂ is CR₅ or N.

Each of x and y, independently, is 0, 1, 2, 3, 4, or 5.

R₃ has the formula:

wherein

Y₁ is alkyl, cycloalkyl, aryl, aralkyl, or alkynyl, wherein Y₁ isoptionally substituted by 0 to 6 R_(n); Y₂ is alkyl, cycloalkyl, aryl,aralkyl, or alkynyl, wherein Y₂ is optionally substituted by 0 to 6R_(n); Y₃ is alkyl, cycloalkyl, aryl, aralkyl, or alkynyl, wherein Y₃ isoptionally substituted by 0 to 6 R_(n); Y₄ is alkyl, cycloalkyl, aryl,aralkyl, or alkynyl, wherein Y₄ is optionally substituted by 0 to 6R_(n); or any two of Y₁, Y₂, and Y₃ are taken together with the N atomto which they are attached to form a 3- to 8-member heterocycleoptionally substituted by 0 to 6 R_(n); or Y₁, Y₂, and Y₃ are all betaken together with the N atom to which they are attached to form abicyclic 5- to 12-member heterocycle optionally substituted by 0 to 6R_(n).

Each R_(n), independently, is H, halo, cyano, hydroxy, amino, alkyl,alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl.

L₃ is a bond, —N(Q)-, —O—, —S—, —(CR₇R₈)_(a)—, —C(O)—, or a combinationof any two of these.

L₄ is a bond, —N(Q)-, —O—, —S—, —(CR₇R₈)_(a)—, —C(O)—, or a combinationof any two of these.

L₅ is a bond, —N(Q)-, —O—, —S—, —(CR₇R₈)_(a)—, —C(O)—, or a combinationof any two of these.

Each occurrence of R₇ and R₈ is, independently, H, halo, cyano, hydroxy,amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl; ortwo R₇ groups on adjacent carbon atoms are taken together to form adouble bond between their respective carbon atoms; or two R₇ groups onadjacent carbon atoms and two R₈ groups on the same adjacent carbonatoms are taken together to form a triple bond between their respectivecarbon atoms.

Each a, independently, is 0, 1, 2, or 3; wherein an R₇ or R₈ substituentfrom any of L₃, L₄, or L₅ is optionally taken with an R₇ or R₈substituent from any of L₃, L₄, or L₅ to form a 3- to 8-membercycloalkyl, heterocyclyl, aryl, or heteroaryl group; and any one of Y₁,Y₂, or Y₃, is optionally taken together with an R₇ or R₈ group from anyof L₃, L₄, and L₅, and atoms to which they are attached, to form a 3- to8-member heterocyclyl group.

Each occurrence of R₅ and R₆ is, independently, H, halo, cyano, hydroxy,amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl.Each Q, independently, is H, alkyl, acyl, cycloalkyl, alkenyl, alkynyl,aryl, heteroaryl or heterocyclyl; and each Q₂, independently, is O, S,N(Q)Q, alkyl or alkoxy.

In some embodiments, the composition can further include a lipid capableof reducing aggregation. The composition can further include a nucleicacid. The nucleic acid can include a chemically modified nucleic acid.The nucleic acid can be 10 to 50 nucleotides long. The nucleic acid canbe an oligonucleotide. The oligonucleotide can be 10 to 50 nucleotideslong. The oligonucleotide can be double stranded or single stranded.More particularly, in some embodiments, the nucleic acid can be siRNA ormRNA. In other embodiments, the nucleic acid can be an antisense nucleicacid, a microRNA, an antimicro RNA, an antagomir, a microRNA inhibitoror an immune stimulatory nucleic acid.

In some embodiments, the sample cells can be in suspension. In somecircumstances, the volume of the sample cells in suspension can be atleast 0.050 L, at least 3 L, at least 25 L or at least 40 L.

In some embodiments, a method for delivering a nucleic acid to samplecells can further include culturing untreated control cells that havenot been exposed to the composition.

In some embodiments, the cell density of the sample cells can increaseafter the sample cells have been exposed to the composition. In somecircumstances, the cell density of the sample cells can increaseexponentially for a period of time after the sample cells have beenexposed to the composition. In some embodiments, the cell density of thesample cells can be greater than or equal to the cell density of theuntreated control cells as measured three days after the sample cellshave been exposed to the composition.

In some embodiments, the sample cell viability can be greater than 90%as measured three days after the sample cells have been exposed to thecomposition.

In some embodiments, a method for delivering a nucleic acid to samplecells can further include measuring a level of a protein in the samplecells and untreated control cells, the protein can be produced from anmRNA that an siRNA delivered into the sample cells was directed against.

In some embodiments, the protein level in the sample cells can be lessthan the protein level in the untreated control cells as measured at oneday after the sample cells have been exposed to the composition. In somecircumstances, the protein level in the sample cells can be less than60% of the protein level in the untreated control cells as measured atone day after the sample cells have been exposed to the composition oras measured at one sample cell doubling time after the sample cells havebeen exposed to the composition. In some circumstances, the proteinlevel in the sample cells can be less than 70% of the protein level inthe untreated control cells as measured at three days after the samplecells have been exposed to the composition or as measured at three timesthe sample cell doubling time after the sample cells have been exposedto the composition. In some circumstances, the protein level in thesample cells can be less than 75% of the protein level in the untreatedcontrol cells as measured at five days after the sample cells have beenexposed to the composition or as measured at five times the sample celldoubling time after the sample cells have been exposed to thecomposition.

In another aspect, a storage-stable composition can include acryoprotectant selected from sucrose, trehalose, glucose,2-hydroxypropyl-α-cyclodextrin, and sorbitol, and a cationic lipidhaving the formula described above. The composition can further includea neutral lipid; a sterol; and/or a lipid capable of reducingaggregation. The composition can include a nucleic acid. Thecryoprotectant can be present at from 5 wt % to 25 wt %, or at from 7 wt% to 15 wt %. The cryoprotectant can include sucrose. The compositioncan be lyophilized, i.e., in a lyophilized state.

In another aspect, a method for reconstituting a storage-stablecomposition can include resuspending the composition in a liquid. It canfurther include adding a lipid and/or a nucleic acid to the resuspendedcomposition. Other features, advantages, and embodiments will beapparent from the description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting relative gene expression in a knockdownexperiment with varying concentrations of siRNA.

FIG. 2 is a graph depicting relative gene expression in a knockdownexperiment as a function of N/P ratio.

FIG. 3 is a graph depicting relative gene expression in a knockdownexperiment with varying concentrations of siRNA.

FIG. 4 is a graph depicting expression knockdown measured usingdifferent transfection compositions.

FIG. 5 is a graph depicting particle sizes of liposomes.

FIGS. 6A-6D are graphs depicting expression knockdown measured usingdifferent transfection compositions.

FIG. 7 is a graph depicting relative gene expression in a knockdownexperiment with varying concentrations of siRNA and varying liposomecompositions.

FIG. 8 is a graph depicting expression knockdown measured usingdifferent transfection compositions.

FIG. 9 is a graph depicting relative gene expression in a knockdownexperiment with varying concentrations of siRNA and varying liposomecompositions.

FIG. 10 is a graph depicting expression knockdown measured usingdifferent transfection compositions.

FIG. 11 is a graph depicting cell viability as a function of lipidconcentration for various lipids.

FIG. 12 is a graph depicting cell viability as a function of lipidconcentration for various lipids.

FIG. 13 is a graph depicting expression knockdown measured usingdifferent transfection compositions.

FIG. 14 is a graph depicting percent GFP signal remaining after a GFPknockdown experiment using different transfection compositions todeliver siRNA directed against GFP mRNA.

FIG. 15 is a graph depicting relative gene expression in a knockdownexperiment using varying concentrations of a transfection reagent, K8.

FIG. 16 is a graph depicting relative gene expression in a knockdownexperiment using varying concentrations of two transfection reagents, K8and P8.

FIG. 17 is a graph depicting cell viability and cell density followingexposure to lipid formulation P8.

FIG. 18 is a graph depicting relative LDH activity as a function oftime.

FIG. 19 is a graph depicting cell viability and cell density followingexposure to lipid formulation P8.

FIG. 20 is a graph depicting relative LDH activity as a function oftime.

DETAILED DESCRIPTION

The present invention is based, in part, upon the discovery of chargedlipids that provide advantages when used in lipid particles for the invivo delivery of a therapeutic agent. In particular, as illustrated bythe accompanying Examples, the present invention provides nucleicacid-lipid particle compositions comprising a charged lipid according tothe present invention. In some embodiments, a composition describedherein provides increased activity of the nucleic acid and/or improvedtolerability of the compositions in vivo, which can result in asignificant increase in therapeutic index as compared to lipid-nucleicacid particle compositions previously described. Additionallycompositions and methods of use are disclosed that can provide foramelioration of the toxicity observed with certain therapeutic nucleicacid-lipid particles.

In certain embodiments, the present invention specifically provides forimproved compositions for the delivery of siRNA molecules. It is shownherein that these compositions are effective in down-regulating theprotein levels and/or mRNA levels of target proteins. Furthermore, it isshown that the activity of these improved compositions is dependent onthe presence of a certain charged lipids and that the molar ratio ofcharged lipid in the formulation can influence activity.

The lipid particles and compositions of the present invention may beused for a variety of purposes, including the delivery of associated orencapsulated therapeutic agents to cells, both in vitro and in vivo.Accordingly, the present invention provides methods of treating diseasesor disorders in a subject in need thereof, by contacting the subjectwith a lipid particle of the present invention associated with asuitable therapeutic agent.

As described herein, the lipid particles of the present invention areparticularly useful for the delivery of nucleic acids, including, e.g.,siRNA molecules and plasmids. Therefore, the lipid particles andcompositions of the present invention may be used to modulate theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid particle of the present inventionassociated with a nucleic acid that reduces target gene expression(e.g., an siRNA) or a nucleic acid that may be used to increaseexpression of a desired protein (e.g., a plasmid encoding the desiredprotein).

Various exemplary embodiments of the charged lipids of the presentinvention, as well as lipid particles and compositions comprising thesame, and their use to deliver therapeutic agents and modulate gene andprotein expression are described in further detail below.

Lipids

The present invention provides novel lipids having certain designfeatures. As shown in FIG. 5, the lipid design features include at leastone of the following: a head group with a quaternary amine, andoptionally, a varying pKa, a cationic, 1°, 2° and 3°, monoamine, di andtriamine, oligoamine/polyamine, a low pKa head groups—imidazoles andpyridine, guanidinium, anionic, zwitterionic and hydrophobic tails caninclude symmetric and/or unsymmetric chains, long and shorter, saturatedand unsaturated chain the back bone includes Backbone glyceride andother acyclic analogs, cyclic, Spiro, bicyclic and polycyclic linkageswith ethers, esters, phosphate and analogs, sulfonate and analogs,disulfides, pH sensitive linkages like acetals and ketals, imines andhydrazones, and oximes.

Lipids can be advantageously used in lipid particles for the in vivodelivery of therapeutic agents to cells. Among such lipids are thosehaving the formula:

Each of R′ or R² is independently a C₁₀ to C₃₀ group having the formula-L^(1a)-(CR^(1a)R^(1b))_(α)-[L^(1b)-(CR^(1a)R^(1b))_(β)]_(γ)-L^(1c)-R^(1c),where: L^(1a) is a bond, —CR^(1a)R^(1b)—, —O—, —CO—, —NR^(1d)—, —S—, ora combination thereof.

Each R^(1a) and each R^(1b), independently, is H; halo; hydroxy; cyano;C₁-C₆ alkyl optionally substituted by halo, hydroxy, or alkoxy; C₃-C₈cycloalkyl optionally substituted by halo, hydroxy, or alkoxy; —OR^(1c);—NR^(1c)R^(1d); aryl; heteroaryl; or heterocyclyl;

Each L^(1b), independently, is a bond, —(CR^(1a)R^(1b))₁₋₂—, —O—, —CO—,—NR^(1d)—, —S—,

or a combination thereof, or has the formula

wherein j, k, and l are each independently 0, 1, 2, or 3, provided thatthe sum of j, k and l is at least 1 and no greater than 8; and R^(1f)and R^(1g) are each independently R^(1b), or adjacent R^(1f) and R^(1g),taken together, are optionally a bond;

or has the formula

wherein j and k are each independently 0, 1, 2, 3, or 4 provided thatthe sum of j and k is at least 1; and R^(1f) and R^(1g) are eachindependently R^(1b), or adjacent R^(1f) and R^(1g), taken together, areoptionally a bond;

or has the formula:

wherein —Ar— is a 6 to 14 membered arylene group optionally substitutedby zero to six R^(1a) groups;

or has the formula:

wherein -Het- is a 3 to 14 membered heterocyclylene or heteroarylenegroup optionally substituted by zero to six R^(1a) groups.

L^(1c) is —(CR^(1a)R^(1b))₁₋₂—, —O—, —CO—, —S—, -,

or a combination thereof.

R^(1c) is H; halo; hydroxy; cyano; C₁-C₆ alkyl optionally substituted byhalo, hydroxy, or alkoxy; C₃-C₈ cycloalkyl optionally substituted byhalo, hydroxy, or alkoxy; aryl; heteroaryl; or heterocyclyl; or R^(1c)has the formula:

R^(1d) is H; halo; hydroxy; cyano; C₁-C₆ alkyl optionally substituted byhalo, hydroxy, or alkoxy; C₃-C₈ cycloalkyl optionally substituted byhalo, hydroxy, or alkoxy; aryl; heteroaryl; or heterocyclyl.

α is 0-6; each β, independently, is 0-6; and γ is 0-6.

represents a connection between L₂ and L₁ which is:

(1) a single bond between one atom of L₂ and one atom of L₁, wherein L₁is C(R_(a)), O, S or N(O); L₂ is —(CR₅R₆)_(x)—, —C(O)—(CR₅R₅)_(x)—,—(CR₅R₆)_(x)—CR₅═CR₅—(CR₅R₆)_(y)—,—C(O)—(CR₅R₆)_(x)—CR₅═CR₅—(CR₅R₆)_(y)—, —S—, ═N—, ═C(R₅)—, —CR₅R₆—O—,—CR₅R₆—N(Q)-, —CR₅R₆—S—, —C(O)N(Q)-, —C(O)O—, —N(Q)C(O)—, —OC (O)—,—C(O)—, or —X—C(R₅)(YR₃)—; wherein X and Y are each, independently,selected from the group consisting of —O—, —S—,

alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —O S(O)(Q₂)O—, and —OP(O)(Q₂)O—.

R_(a) is H, alkyl, alkoxy, —OH, —N(Q)Q, or —SQ.

(2) a double bond between one atom of L₂ and one atom of L₁, wherein L₁is C; L₂ is —(CR₅R₆)_(x)—CR₅═, —C(O)—(CR₅R₆)_(x)—CR₅═, —N(Q)═, —N—,—O—N═, —N(Q)-N═, or —C(O)N(Q)-N═.

(3) a single bond between a first atom of L₂ and a first atom of L₁, anda single bond between a second atom of L₂ and the first atom of L₁,wherein L₁ is C or C(R_(a))—(CR₅R₆)_(x)—C(R_(a)); L₂ has the formula

wherein

X is the first atom of L₂, Y is the second atom of L₂, - - - - -represents a single bond to the first atom of L₁, and X and Y are each,independently, selected from the group consisting of —O—, —S—,

alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —O S(O)(Q₂)O—, and —OP(O)(Q₂)O—; Z₁ and Z₄ are each,independently, —O—, —S—, —CH₂—, —CHR⁵—, or —CR⁵R⁵—; Z₂ is CH or N; Z₃ isCH or N; or Z₂ and Z₃, taken together, are a single C atom; A₁ and A₂are each, independently, —O—, —S—, —CH₂—, —CHR⁵—, or —CR⁵R⁵—.

Each Z is N, C(R₅), or C(R₃).

k is 0, 1, or 2; each m, independently, is 0 to 5; each n,independently, is 0 to 5; where m and n taken together result in a 3, 4,5, 6, 7 or 8 member ring.

(4) a single bond between a first atom of L₂ and a first atom of L₁, anda single bond between the first atom of L₂ and a second atom of L₁,wherein

(A) L₁ has the formula:

wherein

X is the first atom of L₁, Y is the second atom of L₁, - - - - -represents a single bond to the first atom of L₂, and X and Y are each,independently, selected from the group consisting of —O—, —S—, alkylene,—N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O, —OC(O)O—, —OS(O)(Q₂)O—, and —OP(O)(Q₂)O—; T₁ is CH or N; T₂ is CH or N; or T₁ and T₂taken together are C═C.

L₂ is CR₅;

(B) L₁ has the formula:

wherein

X is the first atom of L₁, Y is the second atom of L₁, - - - --represents a single bond to the first atom of L₂, and X and Y are each,independently, selected from the group consisting of —O—, —S—,

alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —O S(O)(Q₂)O—, and —OP(O)(Q₂)O—; T₁ is —CR₅R₆—, —N(Q)-, —O—,or —S—; T₂ is —CR₅R₆—, —N(Q)-, —O—, or —S—.

L₂ is CR₅ or N; each of x and y, independently, is 0, 1, 2, 3, 4, or 5.

R₃ has the formula:

Y₁ is alkyl, cycloalkyl, aryl, aralkyl, or alkynyl, wherein Y₁ isoptionally substituted by 0 to 6 R_(n). Y₂ is alkyl, cycloalkyl, aryl,aralkyl, or alkynyl, wherein Y₂ is optionally substituted by 0 to 6R_(n). Y₃ is alkyl, cycloalkyl, aryl, aralkyl, or alkynyl, wherein Y₃ isoptionally substituted by 0 to 6 R_(n). Y₄ is alkyl, cycloalkyl, aryl,aralkyl, or alkynyl, wherein Y₄ is optionally substituted by 0 to 6R_(n); or any two of Y₁, Y₂, and Y₃ are taken together with the N atomto which they are attached to form a 3- to 8-member heterocycleoptionally substituted by 0 to 6 R_(n); or Y₁, Y₂, and Y₃ are all betaken together with the N atom to which they are attached to form abicyclic 5- to 12-member heterocycle optionally substituted by 0 to 6R_(n).

Each R_(n), independently, is H, halo, cyano, hydroxy, amino, alkyl,alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl. L₃ is a bond,—N(Q)-, —O—, —S—, —(CR₇R₈)_(a)—, —C(O)—, or a combination of any two ofthese. L₄ is a bond, —N(Q)-, —O—, —S—, —(CR₇R₈)_(a)—, —C(O)—, or acombination of any two of these. L₅ is a bond, —N(Q)-, —O—, —S—,—(CR₇R₅)_(a)—, —C(O)—, or a combination of any two of these.

Each occurrence of R₇ and R₈ is, independently, H, halo, cyano, hydroxy,amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl; ortwo R₇ groups on adjacent carbon atoms are taken together to form adouble bond between their respective carbon atoms: or two R₇ groups onadjacent carbon atoms and two R₈ groups on the same adjacent carbonatoms are taken together to form a triple bond between their respectivecarbon atoms.

Each a, independently, is 0, 1, 2, or 3; wherein an R₇ or R₈ substituentfrom any of L₃, L₄, or L₅ is optionally taken with an R₇ or R₈substituent from any of L₃, L₄, or L₅ to form a 3- to 8-membercycloalkyl, heterocyclyl, aryl, or heteroaryl group; and any one of Y₁,Y₂, or Y₃, is optionally taken together with an R₇ or R₈ group from anyof L₃, L₄, and L₅, and atoms to which they are attached, to form a 3- to8-member heterocyclyl group.

Each occurrence of R₅ and R₆ is, independently, H, halo, cyano, hydroxy,amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl.

Each Q, independently, is H, alkyl, acyl, cycloalkyl, alkenyl, alkynyl,aryl, heteroaryl or heterocyclyl.

Each Q₂, independently, is O, S, N(Q)Q, alkyl or alkoxy.

In some embodiments, Y₃ and/or Y₄ is absent, such that the lipid doesnot include a quaternary nitrogen atom.

In one aspect, a compound can have the formula:

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀, alkoxy,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl;

represents a connection between L₂ and L₁ which is:

(1) a single bond between one atom of L₂ and one atom of L₁, wherein

-   -   L₁ is C(R_(x)) or N;    -   L₂ is —CR₅R₆—, —O—, —S—, —N(Q)-, ═C(R₅)—, —C(O)N(Q)-, —C(O)O—,        —N(Q)C(O)—, —OC(O)—, or —C(O)—;

(2) a double bond between one atom of L₂ and one atom of L₁; wherein

-   -   L₁ is C;    -   L₂ is —CR₅═, —N(Q)═, —N—, —O—N═, —N(Q)-N═, or —C(O)N(Q)-N═;

(3) a single bond between a first atom of L₂ and a first atom of L₁, anda single bond between a second atom of L₂ and the first atom of L₁,wherein

-   -   L₁ is C;    -   L₂ has the formula

wherein

-   -   X is the first atom of L₂, Y is the second atom of L₂, - - - -        -represents a single bond to the first atom of L₁, and X and Y        are each, independently, selected from the group consisting of        —O—, —S—,        alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—,        —C(O)O, —OC(O)O—, —O S(O)(Q₂)O—, and —OP(O)(Q₂)O—;    -   Z₁ and Z₄ are each, independently, —O—, —S—, —CH₂—, —CHR⁵—, or        —CR⁵R⁵—;    -   Z₂ is CH or N;    -   Z₃ is CH or N;    -   or Z₂ and Z₃, taken together, are a single C atom;    -   A₁ and A₂ are each, independently, —O—, —S—, —CH₂—, —CHR⁵—, or        —CR⁵R⁵—;    -   each Z is N, C(R₅), or C(R₃);    -   k is 0, 1, or 2;    -   each m, independently, is 0 to 5;    -   each n, independently, is 0 to 5;    -   where m and n taken together result in a 3, 4, 5, 6, 7 or 8        member ring;

(4) a single bond between a first atom of L₂ and a first atom of L₁, anda single bond between the first atom of L₂ and a second atom of L₁,wherein

-   -   (A) L₁ has the formula:

-   -    wherein        -   X is the first atom of L₁, - - - - - is the second atom of            L₁, represents a single bond to the first atom of L₂, and X            and Y are each, independently, selected from the group            consisting of —O—, —S—,        -    alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-,            —N(Q)C(O)O—, —C(O)O, —OC(O)O—, —O S(O)(Q₂)O—, and            —OP(O)(Q₂)O—;        -   T₁ is CH or N;        -   T₂ is CH or N;        -   or T₁ and T₂ taken together are C═C;        -   L₂ is CR₅; or    -   (B) L₁ has the formula:

-   -    wherein        -   X is the first atom of L₁, Y is the second atom of            L₁, - - - - -represents a single bond to the first atom of            L₂, and X and Y are each, independently, selected from the            group consisting of —O—, —S—, alkylene, —N(Q)-, —C(O)—,            —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O, —OC(O)O—,            —OS(O)(Q₂)O—, and —OP(O)(Q₂)O—;        -   T₁ is —CR₅R₅—, —N(Q)-, —O—, or —S—;        -   T₂ is —CR₅R₅—, —N(Q)-, —O—, or —S—;        -   L₂ is CR₅ or N;

R₃ has the formula:

-   -   wherein

each of Y₁, Y₂, Y₃, and Y₄, independently, is alkyl, cycloalkyl, aryl,aralkyl, or alkynyl; or

any two of Y₁, Y₂, and Y₃ are taken together with the N atom to whichthey are attached to form a 3- to 8-member heterocycle; or

Y₁, Y₂, and Y₃ are all be taken together with the N atom to which theyare attached to form a bicyclic 5- to 12-member heterocycle;

each R_(n), independently, is H, halo, cyano, hydroxy, amino, alkyl,alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl;

L₃ is a bond, —N(Q)-, —O—, —S—, —(CR₅R₆)₄—, —C(O)—, or a combination ofany two of these;

L₄ is a bond, —N(Q)-, —O—, —S—, —(CR₅R₆)_(a)—, —C(O)—, or a combinationof any two of these;

L₅ is a bond, —N(Q)-, —O—, —S—, —(CR₅R₆)_(a)—, —C(O)—, or a combinationof any two of these;

each occurrence of R₅ and R₆ is, independently, H, halo, cyano, hydroxy,amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl; ortwo R₅ groups on adjacent carbon atoms are taken together to form adouble bond between their respective carbon atoms; or two R₅ groups onadjacent carbon atoms and two R₆ groups on the same adjacent carbonatoms are taken together to form a triple bond between their respectivecarbon atoms;

each a, independently, is 0, 1, 2, or 3;

wherein

-   -   an R₅ or R₆ substituent from any of L₃, L₄, or L₅ is optionally        taken with an R₅ or R₆ substituent from any of L₃, L₄, or L₅ to        form a 3- to 8-member cycloalkyl, heterocyclyl, aryl, or        heteroaryl group; and    -   any one of Y₁, Y₂, or Y₃, is optionally taken together with an        R₅ or R₆ group from any of L₃, L₄, and L₅, and atoms to which        they are attached, to form a 3- to 8-member heterocyclyl group;

each Q, independently, is H, alkyl, acyl, cycloalkyl, alkenyl, alkynyl,aryl, heteroaryl or heterocyclyl; and

each Q₂, independently, is O, S, N(Q)(Q), alkyl or alkoxy.

In some embodiments,

represents a connection between L₂ and L₁ which is a single bond betweenone atom of L₂ and one atom of L₁, wherein L₁ is C(R_(x)), O, S or N(Q);and L₂ is —CR₅R₆—, —O—, —S—, ═C(R_(s))—, —C(O)N(Q)-, —C(O)O—,—N(Q)C(O)—, —OC(O)—, or —C(O)—.

In another aspect, a compound having formula I, XIII, XV, XVII, XXXIII,or XXXV:

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkoxy,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl;

R₃ is independently for each occurrence H, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, optionally substituted alkylheterocycle, optionallysubstituted heterocyclealkyl, optionally substituted alkylphosphate,optionally substituted phosphoalkyl, optionally substitutedalkylphosphorothioate, optionally substituted phosphorothioalkyl,optionally substituted alkylphosphorodithioate, optionally substitutedphosphorodithioalkyl, optionally substituted alkylphosphonate,optionally substituted phosphonoalkyl, optionally substituted amino,optionally substituted alkylamino, optionally substituteddi(alkyl)amino, optionally substituted aminoalkyl, optionallysubstituted alkylaminoalkyl, optionally substituted di(alkyl)aminoalkyl,optionally substituted hydroxyalkyl, optionally substituted polyethyleneglycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),optionally substituted heteroaryl, or optionally substitutedheterocycle;

at least one R₃ includes a quaternary amine;

X and Y are each independently —O—, —S—,

alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —O S(O)(Q₂)O—, or —OP(O)(Q₂)O—;

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl,or ω-thiophosphoalkyl;

Q₂ is independently for each occurrence O, S, N(Q)(Q), alkyl or alkoxy;

A₁, A₂, A₃, A₄, A₅ and A₆ are each

independently —O—, —S—, —CH₂—, —CHR⁵—, —CR⁵R⁵—;

A₈ is independently for each occurrence —CH₂—, —CHR⁵—, —CR⁵R⁵—;

E and F are each independently for each

occurrence —CH₂—, —O—, —S—, —SS—, —CO—, —C(O)O—, —C(O)N(R′)—,—OC(O)N(R′)—, —N(R′)C(O)N(R″)—, —C(O)—N(R′)—N═C(R′″)—; —N(R′)—N═C(R″)—,—O—N═C(R″)—, —C(S)O—, —C(S)N(R′)—, —OC(S)N(R′)—, —N(R′)C(S)N(R″)—,—C(S)—N(R′)—N═C(R′″); —S—N═C(R″); —C(O)S—, —SC(O) N(R′)—, —OC(O)—,—N(R′)C(O)—, —N(R′)C(O)O—, —C(R′″)═N—N(R′)—; —C(R′″)═N—N(R′)—C(O)—,—C(R′″)═N—O—, —OC(S)—, —SC(O)—, —N(R′)C(S)—, —N(R′)C(S)O—, —N(R′)C(O)S—,—C(R′″)═N—N(R′)—C(S)—, —C(R′″)═N—S—, C[═N(R′)]O,

C[═N(R′)]N(R″), —OC[═N(R′)]—, —N(R″)C[═N(R′)]N(R′″)—, —N(R″)C[═N(R′)]—,

arylene, heteroarylene, cycloalkylene, or heterocyclylene;

Z is N or C(R₃);

Z′ is —O—, —S—, —N(Q)-, or alkylene;

each R′, R″, and R″, independently, is H, alkyl, alkyl, heteroalkyl,aralkyl, cyclic alkyl, or heterocyclyl;

R⁵ is H, halo, cyano, hydroxy, amino, optionally substituted alkyl,optionally substituted alkoxy, or optionally substituted cycloalkyl;

i and j are each independently 0-10; and

a and b are each independently 0-2.

In another aspect, a compound can be selected from the group consistingof:

In another aspect, a composition can include a compound as describedabove, a neutral lipid, and a sterol. The composition can furtherinclude a nucleic acid. The nucleic acid can be RNA.

In one embodiment, E is O(CO), (CO)O, OC(O)N(R′), or N(R′)C(O)O.

In another aspect, the lipid has one of the following structures, saltsor isomers thereof:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkoxy,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl.

R₃ is independently for each occurrence H, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, optionally substituted alkylheterocycle, optionallysubstituted heterocyclealkyl, optionally substituted alkylphosphate,optionally substituted phosphoalkyl, optionally substitutedalkylphosphorothioate, optionally substituted phosphorothioalkyl,optionally substituted alkylphosphorodithioate, optionally substitutedphosphorodithioalkyl, optionally substituted alkylphosphonate,optionally substituted phosphonoalkyl, optionally substituted amino,optionally substituted alkylamino, optionally substituteddi(alkyl)amino, optionally substituted aminoalkyl, optionallysubstituted alkylaminoalkyl, optionally substituted di(alkyl)aminoalkyl,optionally substituted hydroxyalkyl, optionally substituted polyethyleneglycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),optionally substituted heteroaryl, or optionally substitutedheterocycle.

At least one R₃ includes a quaternary amine.

X and Y are each independently —O—, —S—,

alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —O S(O)(Q₂)O—, or —OP(O)(Q₂)O—.

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl,or ω-thiophosphoalkyl.

Q₂ is independently for each occurrence O, S, N(Q)(Q), alkyl or alkoxy.

A₁, A₂, A₃, A₄, A₅ and A₆ are each independently —O—, —S—, —CHR⁵—,—CR⁵R⁵—.

A₈ is independently for each occurrence —CH₂—, —CHR⁵—, —CR⁵R⁵—.

E and F are each independently for each

occurrence —CH₂—, —O—, —S—, —SS—, —CO—, —C(O)O—, —C(O)N(R′)—,—OC(O)N(R′)—, —N(R′)C(O)N(R″)—, —C(O)—N(R′)—N═C(R′″)—; —N(R′)—N═C(R″)—,—O—N═C(R″)—, —C(S)O—, —C(S)N(R′)—, —OC(S)N(R′)—, —N(R′)C(S)N(R″)—,—C(S)—N(R′)—N═C(R′″); —S—N═C(R″); —C(O)S—, —SC(O) N(R′)—, —OC(O)—,—N(R′)C(O)—, —N(R′)C(O)O—, —C(R′″)═N—N(R′)—; —C(R′″)═N—N(R′)—C(O)—,—C(R″″)═N—O—, —OC(S)—, —SC(O)—, —N(R′)C(S)—, —N(R′)C(S)O—, —N(R′)C(O)S—,—C(R′″)═N—N(R′)—C(S)—, —C(R′″)═N—S—, C[═N(R′)]O,

C[═N(R′)]N(R″), —OC[═N(R′)]—, —N(R″)C[═N(R′)]N(R′″)—, —N(R″)C[═N(R′)]—,

arylene, heteroarylene, cycloalkylene, or heterocyclylene.

Z is N or C(R₃).

Z′ is —O—, —S—, —N(Q)-, or alkylene.

Each R′, R″, and R′″, independently, is H, alkyl, alkyl, heteroalkyl,aralkyl, cyclic alkyl, or heterocyclyl.

R⁵ is H, halo, cyano, hydroxy, amino, optionally substituted alkyl,optionally substituted alkoxy, or optionally substituted cycloalkyl.

i and j are each independently 0-10.

a and b are each independently 0-2.

In some circumstances, R₃ is ω-(substituted)aminoalkyl. The ω-aminogroup can be a quaternary amine. Examples quaternaryω-(substituted)aminoalkyl groups include 2-(trimethylamino)ethyl,3-(triisopropylamino)propyl, or3-(N-methyl-N-ethyl-N-isopropylamino)-1-methylpropyl.

In some circumstances, R₃ has the formula:

where each of Y₁, Y₂, and Y₃, is independently, alkyl, cycloalkyl, aryl,aralkyl, or alkynyl. For example, each of Y₁, Y₂, and Y₃, isindependently C₁-C₆ alkyl or C₃-C₆ cycloalkyl, C₁-C₄ alkyl, or C₁-C₃alkyl.

Any two of Y₁, Y₂, and Y₃ can be taken together with the N atom to whichthey are attached to form a 3- to 8-member heterocycle. For example, Y₁,and Y₂ can be taken together with the N atom to which they are attachedto form a pyrrolidine, a pyrrole, an oxazole, an imidazole, a pyridine,a piperidine, or other N-containing heterocycles. In some cases, Y₁, Y₂,and Y₃ can all be taken together with the N atom to which they areattached to form a bicyclic 5- to 12-member heterocycle. For example,Y₁, Y₂, and Y₃ can all be taken together the N atom to which they areattached to form a quinuclidine, a tropane, a1,4-diazabicyclo[2.2.2]octane, or other bicyclic heterocycles.

Each Y₁, Y₂, Y₃, independently, can be optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted aryl,optionally substituted aralkyl, or optionally substituted alkynyl. Whentwo or more of Y₁, Y₂, and Y₃ are taken together with the N atom towhich they are attached to form a heterocyclic group, the heterocyclicgroup can be optionally substituted.

L₃ is a bond, —N(Q)-, —O—, —S—, —(CR₅R₆)_(a)—, —C(O)—, or a combinationof any two of these;

L₄ is a bond, —N(Q)-, —O—, —S—, —(CR₅R₆)_(a)—, —C(O)—, or a combinationof any two of these;

L₅ is a bond, —N(Q)-, —O—, —S—, —(CR₅R₆)_(a)—, —C(O)—, or a combinationof any two of these;

Each of R₅ and R₆ is, independently, H, halo, cyano, hydroxy, amino,alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl. Two R₅groups on adjacent carbon atoms can be taken together to form a doublebond between their respective carbon atoms. Two R₅ groups on adjacentcarbon atoms and two R₆ groups on the same adjacent carbon atoms can betaken together to form a triple bond between their respective carbonatoms.

Each R₅ and R₆, independently, can be optionally substituted alkyl,optionally substituted alkoxy, optionally substituted cycloalkyl,optionally substituted aryl, optionally substituted heteroaryl, oroptionally substituted heterocyclyl.

Each a, independently, is 0, 1, 2, or 3.

In some cases, an R₅ or R₆ substituent from L₃ can be taken with an R₅or R₆ substituent from L₄ to form a 3- to 8-member cycloalkyl orheterocycle group. Similarly, an R₅ or R₆ substituent from L₃ can betaken with an R₅ or R₆ substituent from L₅ to form a 3- to 8-membercycloalkyl or heterocycle group; or an R₅ or R₆ substituent from L₄ canbe taken with an R₅ or R₆ substituent from L₅ to form a 3- to 8-membercycloalkyl or heterocycle group. A cycloalkyl group or heterocycle groupformed by R₅ or R₆ substituents from L₃, L₄, or L₅ can be optionallysubstituted. By way of illustration only, one exemplary R₃ group havingthis structural feature includes(5-(2-(N,N-diethyl-N-methyeamino)ethyl-4-methyl-1,3-dioxan-2-yl)methyl.

In some cases, Y₁, Y₂, or Y₃ can be taken together with an R₅ or R₆group from any of L₃, L₄, and L₅, and atoms to which they are attached,to form a 3- to 8-member heterocycle. By way of illustration only, someexemplary R₃ groups having this structural feature include(N,N-dimethylpyrrolidin-2-yl)methyl, and2-(N-methyl-N-ethylpiperidin-4-yl)ethyl.

In one embodiment, X and Y can be independently —O—, —S—, alkylene, or—N(Q)-.

It has been found that charged lipids comprising unsaturated alkylchains are particularly useful for forming lipid nucleic acid particleswith increased membrane fluidity. In one embodiment, at least one of R₁or R₂ comprises at least one, at least two or at least three sites ofunsaturation, e.g. double bond or triple bond.

In one embodiment, only one of R₁ or R₂ comprises at least one, at leasttwo or at least three sites of unsaturation.

In one embodiment, R₁ and R₂ both comprise at least one, at least two orat least three sites of unsaturation.

In one embodiment, R₁ and R₂ comprise different numbers of unsaturation,e.g., one of R₁ and R₂ has one site of unsaturation and the other hastwo or three sites of unsaturation.

In one embodiment, R₁ and R₂ both comprise the same number ofunsaturation sites.

In one embodiment, R₁ and R₂ comprise different types of unsaturation,e.g. unsaturation in one of R₁ and R₂ is double bond and in the otherunsaturation is triple bond.

In one embodiment, R₁ and R₂ both comprise the same type ofunsaturation, e.g. double bond or triple bond.

In one embodiment, at least one of R₁ or R₂ comprises at least onedouble bond and at least one triple bond.

In one embodiment, only one of R₁ or R₂ comprises at least one doublebond and at least one triple bond.

In one embodiment, R₁ and R₂ both comprise at least one double bond andat least one triple bond.

In one embodiment, R₁ and R₂ are both same, e.g. R₁ and R₂ are bothlinoleyl (C18) or R₁ and R₂ are both heptadeca-9-enyl.

In one embodiment, R₁ and R₂ are different from each other.

In one embodiment, at least one of R₁ and R₂ is cholesterol.

In one embodiment, at least one of R₁ or R₂ comprises at least onemethylene group where one or both H atoms are replaced by F, e.g.fluoromethylene or difluoromethylene. In one embodiment, both R₁ and R₂comprise at least one methylene group with one or two H replaced by F,e.g. fluoromethylene or difluoromethylene.

In one embodiment, only one of R₁ and R₂ comprises at least onemethylene group with one or both H replaced by F.

In one embodiment, at least one of R₁ or R₂ terminates in fluoromethyl,difluormethyl, or trifluoromethyl. In one embodiment, both R₁ and R₂terminate in fluoromethyl, difluormethyl, or trifluoromethyl.

In one embodiment, at least one of R₁ or R₂ is—(CF₂)_(y)—Z″—(CH₂)_(y)—CH₃, wherein each y is independently 1-10 and Z″is O, S or N(Q).

In one embodiment, both of R₁ and R₂ are —(CF₂)_(y)—Z″—(CH₂)_(y)—CH₃,wherein each y is independently 1-10 and Z″ is O, S or N(Q).

In one embodiment, at least one of R₁ or R₂ is—(CH₂)_(y)—Z″—(CF₂)_(y)—CF₃, wherein each y is independently 1-10 and Z″is O, S or N(Q).

In one embodiment, both of R₁ and R₂ are —(CH₂)_(y)—Z″—(CF₂)_(y)—CF₃,wherein each y is independently 1-10 and Z″ is O, S or N(Q).

In one embodiment, at least one of R₁ or R₂ is —(CF₂)_(y)—(CF₂)_(y)—CF₃,wherein each y is independently 1-10.

In one embodiment, both of R₁ and R₂ are —(CF₂)_(y)—(CF₂)_(y)—CF₃,wherein each y is independently 1-10.

In some embodiments, R₁ and R₂ are, independently, selected from thegroup consisting of lineolyl, γ-linoenyl, n-octadecanyl, n-decanyl,n-dodecanyl, and 9-methyloctadecanyl. In some embodiments the lipid canhave (R₁, R₂) selected from the group consisting of (lineolyl,lineolyl), γ-linoenyl, γ-linoenyl), (lineolyl, n-octadecanyl),(lineolyl, n-decanyl), (lineolyl, n-dodecanyl), and(9-methyloctadecanyl, 9-methyloctadecanyl).

In one embodiment, when Z is C(R₃), at least one R₃ is ω-aminoalkyl orω-(substituted)aminoalkyl.

In one embodiment, when Z′ is O, S or alkyl, at least one R₃ isω-aminoalkyl or ω-(substituted)aminoalkyl.

In one embodiment, the lipid is a racemic mixture.

In one embodiment, the lipid is enriched in one diastereomer, e.g. thelipid has at least 95%, at least 90%, at least 80% or at least 70%diastereomeric excess.

In one embodiment, the lipid is enriched in one enantiomer, e.g. thelipid has at least 95%, at least 90%, at least 80% or at least 70%enantiomer excess.

In one embodiment, the lipid is chirally pure, e.g. is a single opticalisomer.

In one embodiment, the lipid is enriched for one optical isomer.

Where a double bond is present (e.g., a carbon-carbon double bond orcarbon-nitrogen double bond), there can be isomerism in theconfiguration about the double bond (i.e. cis/trans or E/Z isomerism).Where the configuration of a double bond is illustrated in a chemicalstructure, it is understood that the corresponding isomer can also bepresent. The amount of isomer present can vary, depending on therelative stabilities of the isomers and the energy required to convertbetween the isomers. Accordingly, some double bonds are, for practicalpurposes, present in only a single configuration, whereas others (e.g.,where the relative stabilities are similar and the energy of conversionlow) may be present as inseparable equilibrium mixture ofconfigurations.

In another aspect, the invention features a compound of formula XXXIVa,XXXIVb, XXXIVc, XXXIVd, or XXXIVe, salts or isomers thereof:

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl, oroptionally substituted C₁₀-C₃₀ alkynyl;

R₃ is defined as above.

n is 1, 2, or 3.

In some embodiments, R₃ is optionally substituted heterocyclealkyl,optionally substituted amino, optionally substituted alkylamino,optionally substituted di(alkyl)amino, optionally substitutedaminoalkyl, optionally substituted alkylaminoalkyl, optionallysubstituted di(alkyl)aminoalkyl, or optionally substituted heterocycle.

In one aspect, the lipid is a compound of formula

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl, oroptionally substituted C₁₀-C₃₀ alkynyl.

At least one of R₃ and R_(3′) includes a quaternary amine.

R₃ and R_(3′) are independently for each occurrence defined as R₃ above;

or R₃ and R_(3′) can be taken together with the atoms to which they areattached to form an optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl or optionallysubstituted heteroaryl; each of which is substituted with 0-4occurrences of R₄;

each R₄ is independently selected from optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, optionally substituted amino, optionally substitutedalkylamino, optionally substituted di(alkyl)amino, optionallysubstituted aminoalkyl, optionally substituted alkylaminoalkyl,optionally substituted di(alkyl)aminoalkyl, optionally substitutedhydroxyalkyl, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocycle;

X and Y are each independently —O—, —S—, alkylene, or —N(Q)-;

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl,or ω-thiophosphoalkyl;

A₁ and A₂ are each independently —O—, —S—, or —CR⁵R⁵—; and

R⁵ is H, halo, cyano, hydroxy, amino, optionally substituted alkyl,optionally substituted alkoxy, or optionally substituted cycloalkyl; and

Z and Z′ are each independently selected from —O—, —S—, —N(Q)-, alkyleneor absent; and

a and b are each independently 0-2.

In some embodiments, X and Y are each independently O.

In some embodiments, the sum of a and b is 1, 2, or 3.

In some embodiments, A₁ and A₂ are each independently —CR⁵R⁵—.

In some embodiments, Z and Z′ are each a bond.

In some embodiments, R₃ and R_(3′) can be taken together with the atomsto which they are attached to form an optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl or optionally substituted heteroaryl.

In some embodiments, R₃ and R₃′ can be taken together with the atoms towhich they are attached to form an optionally substituted carbocyclyl(e.g., optionally substituted with amino, alkylamino, or dialkylamino).

In some embodiments, R₃ and R_(3′), can be taken together with the atomsto which they are attached to form an optionally substitutedheterocyclyl (e.g., a nitrogen containing heterocyclyl).

In some embodiments, R₃ and R_(3′) are taken together to form acarbocyclic ring (e.g., cyclohexyl) substituted with 0-3 occurrence ofR₄.

In some embodiments, R₃ and R_(3′) are taken together to form aheterocyclic ring (e.g., piperidine) substituted with 0-3 occurrences ofR₄.

In some embodiments, each R₄ is independently selected from optionallyoptionally substituted amino, optionally substituted alkylamino,optionally substituted di(alkyl)amino, optionally substitutedaminoalkyl, optionally substituted alkylaminoalkyl, optionallysubstituted di(alkyl)aminoalkyl, and optionally substitutedhydroxyalkyl.

In one aspect, the lipid is a compound of formula XXXIX, salts orisomers thereof:

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀acyl.

R₃ is defined as above.

X and Y are each independently O, C(O)O, S, alkyl or N(Q);

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkylor ω-thiophosphoalkyl.

In one aspect, the lipid is a compound of formula XXXIII, salts orisomers thereof

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀acyl;

Eis —CH₂—, —O—, —S—, —SS—, —CO—, —C(O)O—, —C(O)N(R′)—, —OC(O)N(R′)—,—N(R′)C(O)N(R″)—, —C(O)—N(R′)—N═C(R′″)—; —N(R′)—N═C(R″)—, —O—N═C(R″)—,—C(S)O—, —C(S)N(R′)—, —OC(S)N(R′)—, —N(R′)C(S)N(R″)—,—C(S)—N(R′)—N═C(R′″); —S—N═C(R″); —C(O)S—, —SC(O) N(R′)—, —OC(O)—,—N(R′)C(O)—, —N(R′)C(O)O—, —C(R′″)═N—N(R′)—; —C(R′″)═N—N(R′)—C(O)—,—C(R′″)═N—O—, —OC(S)—, —SC(O)—, —N(R′)C(S)—, —N(R′)C(S)O—, —N(R′)C(O)S—,—C(R′″)═N—N(R′)—C(S)—, —C(R′″)═N—S—, C[═N(R′)]O,

C[═N(R′)]N(R″), —OC[═N(R′)]—, —N(R″)C[═N(R′)]N(R′″)—, —N(R″)C[═N(R′)]—,

arylene, heteroarylene, cycloalkylene, or heterocyclylene.

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalky, ω-phosphoalkyl orω-thiophosphoalkyl.

In one embodiment, R₁ and R₂ are each independently for each occurrenceoptionally substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀alkoxy, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl.

In one embodiment, the lipid is a compound of formula XXXIII, providedthat when E is —C(O)O— and R³ is

R¹ and R² are not both linoleyl.

In one embodiment, the invention features a lipid of formula)(XXVIII:

wherein

Eis —CH₂—, —O—, —S—, —SS—, —CO—, —C(O)O—, —C(O)N(R′)—, —OC(O)N(R′)—,—N(R′)C(O)N(R″)—, —C(O)—N(R′)—N═C(R′″)—; —N(R′)—N═C(R″)—, —O—N═C(R″)—,—C(S)O—, —C(S)N(R′)—, —OC(S)N(R′)—, —N(R′)C(S)N(R″)—,—C(S)—N(R¹)—N═C(R′″); —S—N═C(R″); —C(O)S—, —SC(O)N(R′)—, —OC(O)—,—N(R′)C(O)—, —N(R′)C(O)O—, —C(R′″)═N—N(R′)—; —C(R′″)═N—N(R′)′C(O)′,—C(R′″)═N—O—, —OC(S)—, —SC(O)—, —N(R′)C(S)—, —N(R′)C(S)O—, —N(R′)C(O)S—,—C(R′″)═N—N(R′)—C(S)—, —C(R′″)═N—S—, C[═N(R′)]O,

C[═N(R′)]N(R″), —OC[═N(R′)]—, —N(R″)C[═N(R′)]N(R′″)—, —N(R″)C[═N(R′)]—,

arylene, heteroarylene, cycloalkylene, or heterocyclylene.

R₃ has the formula:

Y₁ is alkyl, cycloalkyl, aryl, aralkyl, or alkynyl, wherein Y₁ isoptionally substituted by 0 to 6 R_(n). Y₂ is alkyl, cycloalkyl, aryl,aralkyl, or alkynyl, wherein Y₂ is optionally substituted by 0 to 6R_(n). Y₃ is alkyl, cycloalkyl, aryl, aralkyl, or alkynyl, wherein Y₃ isoptionally substituted by 0 to 6 R_(n). Y₄ is alkyl, cycloalkyl, aryl,aralkyl, or alkynyl, wherein Y₄ is optionally substituted by 0 to 6R_(n); or any two of Y₁, Y₂, and Y₃ are taken together with the N atomto which they are attached to form a 3- to 8-member heterocycleoptionally substituted by 0 to 6 R_(n); or Y₁, Y₂, and Y₃ are all betaken together with the N atom to which they are attached to form abicyclic 5- to 12-member heterocycle optionally substituted by 0 to 6R_(n).

Each R_(n), independently, is H, halo, cyano, hydroxy, amino, alkyl,alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl. L₃ is a bond,—N(Q)-, —O—, —S—, —(CR₇R₈)_(a)—, —C(O)—, or a combination of any two ofthese. L₄ is a bond, —N(Q)-, —O—, —S—, —(CR₇R₈)_(a), —C(O)—, or acombination of any two of these. L₅ is a bond, —N(Q)-, —O—, —S—,—(CR₇R₈)_(a)—, —C(O)—, or a combination of any two of these.

Each occurrence of R₇ and R₈ is, independently, H, halo, cyano, hydroxy,amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl; ortwo R₇ groups on adjacent carbon atoms are taken together to form adouble bond between their respective carbon atoms; or two R₇ groups onadjacent carbon atoms and two R₃ groups on the same adjacent carbonatoms are taken together to form a triple bond between their respectivecarbon atoms.

Each a, independently, is 0, 1, 2, or 3; wherein an R₇ or R₈ substituentfrom any of L₃, L₄, or L₅ is optionally taken with an R₇ or R₈substituent from any of L₃, L₄, or L₅ to form a 3- to 8-membercycloalkyl, heterocyclyl, aryl, or heteroaryl group; and any one of Y₁,Y₂, or Y₃, is optionally taken together with an R₇ or R₈ group from anyof L₃, L₄, and L₅, and atoms to which they are attached, to form a 3- to8-member heterocyclyl group.

Each occurrence of R₅ and R₆ is, independently, H, halo, cyano, hydroxy,amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, or heterocyclyl.

Each Q, independently, is H, alkyl, acyl, cycloalkyl, alkenyl, alkynyl,aryl, heteroaryl or heterocyclyl.

Each Q₂, independently, is O, S, N(Q)Q, alkyl or alkoxy.

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl,or ω-thiophosphoalkyl.

R₁ and R₂ and R_(x) are each independently for each occurrence H,optionally substituted C₁-C₁₀ alkyl, optionally substituted C₁₀-C₃₀alkyl, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀ acyl. In someembodiments, at least one of R₁, R₂ and R_(x) is not H.

In some embodiments, at least two of R₁, R₂ and R_(x) is not H.

R₃ is defined as above.

n is 0, 1, 2, or 3.

In one embodiment, where the lipid is a compound of formula XXXVIII,when E is C(O)O, R³ is

and one of R₁, R₂, or R_(x) is H, then the remaining of R₁, R₂ or R_(x)are not both linoleyl.

In some embodiments, each of R₁ and R₂ is independently for eachoccurrence optionally substituted C₁₀-C₃₀ alkyl, optionally substitutedC₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ acyl.

In some embodiments, R_(x) is H or optionally substituted C₁-C₁₀ alkyl.

In some embodiments, R_(x) is optionally substituted C₁₀-C₁₀ alkyl,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₁₀alkynyl, optionally substituted C₁₀-C₃₀ acyl.

The present invention comprises of synthesis of lipids described hereinin racemic as well as in optically pure form.

In one aspect, a lipid has formula LX, LXI, LXII, LXIII, LXIV, LXV,LXVI, LXVII, LXVIII, LXIX, LXX, LXXI, LXXII, or LXXIII:

wherein:

X and Y are each independently —O—, —S—, —CH₂—, or —N(Q₃)-; where Q isH, Me, Et, or —(CH₂)_(r)—N(Q₃)(Q₄);

Z is N, CH, C(Me), C(Et);

Q₁ is O or S;

Q₂ is O or S;

Each of A₁ and A₂, independently, are CH₂, CHF, or CF₂;

m, n, p and q are each independently 0 to 5.

In formulas LX, LXI, LXII, LXIII, LXIV, LXV, LXVI, LXVII, LXVIH, LXIX,LXX, LXXI, LXXII, and LXXIII, R₁, R₂ and R₄ are each independentlyselected from the group consisting of alkyl groups having about 10 to 30carbon atoms, wherein R₁, R₂ and R₄ independently comprises of: fullysaturated alkyl chain, at least one double bond, at least one triplebond, at least one hetero atom, at least one CF₂, at least one CHF or atleast one perfluoroalkylated chain. CF₂/CHF could be on the lipid anchoror on the core.

R₃ is defined as above.

In one embodiment, the lipid can be a compound having the formula:

or having the formula:

or a mixture thereof.

Each R_(a), independently, is absent, H, alkyl, or cycloalkyl. In oneembodiment, R_(a) is alkyl or cycloalkyl for no more than twooccurrences. In one embodiment, R_(a) is alkyl or cycloalkyl for no morethan one occurrence.

In one embodiment, R, for at least 3 occurrences, is

In one embodiment, Y is O or NR⁴. In one embodiment, Y is O. In oneembodiment, Y is O for each occurrence. In one embodiment, R¹ is H. Inone embodiment, R¹ is H for each occurrence.

In one embodiment, R¹ is

wherein R³ alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, orheteroalkynyl, each of which is optionally substituted with one or moresubstituent (e.g., a hydrophilic substituent). In one embodiment, R¹ is

and R³ alkyl optionally substituted with one or more substituent (e.g.,a hydrophilic substituent). In one embodiment, R³ is substituted with—OH.

In one embodiment, R¹ is R³,

wherein R³ alkyl is optionally substituted with one or more substituent.In one embodiment, R³ is substituted with a hydrophilic substituent. R⁴,for each occurrence is independently H alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, or heteroalkynyl; each of which isoptionally substituted with one or more substituent. In one embodiment,R³ is substituted with —OH. In one embodiment, R² is alkyl, alkenyl, oralkynyl. In one embodiment, R² is alkyl (e.g., C₆-C₁₈ alkyl, e.g.,C₈-C₁₂ alkyl, e.g., C₁₀ alkyl).

In one embodiment, R for at least 3 (e.g., at least 4 or 5) occurrencesis

In one embodiment, R² is alkyl (e.g., C₆-C₁₈ alkyl, e.g., C₈-C₁₂ alkyl,e.g., C₁₀ alkyl). In one embodiment, R for at least 1 occurrence (e.g.,1 or 2 occurrences) is H.

The lipid can be a compound having the formula:

or a compound having the formula:

or a mixture thereof.

In one embodiment, no more than two instances of R_(a) are alkyl orcycloalkyl. In one embodiment, no more than one instance of R_(a) arealkyl or cycloalkyl. In one embodiment, one or two instances of R_(a)are methyl, and the remaining instances of R_(a) are each absent or H.

In some embodiments, the lipid can be a quaternary lipid derived fromthe compounds disclosed in Akinc, A., et al., “Development oflipidoid-siRNA formulations for systemic delivery of RNAi therapeutics,”Nat. Biotechnol. 26, (2008), 561-569; Love, K. T., et al., “Lipid-likematerials for low-dose, in vivo gene silencing,” PNAS 107, 5, (2010),1864-1869; or Mahon, K. P., et al., “Combinatorial approach to determinefunctional group effects on lipidoid-mediated siRNA delivery,” BioconjugChem. 2010 Aug. 18; 21(8):1448-54; each of which is incorporated byreference in its entirety.

For example, the lipid can have one of the following formulas:

where R is

where x is 3-15, and each R^(a), independently, is absent, H, alkyl, orcycloalkyl. In some cases, R^(a) is alkyl or cycloalkyl for no more thantwo occurrences, or R^(a) can be alkyl or cycloalkyl for no more thanone occurrence. In some cases, R^(a) is methyl.

In another example, the lipid can have one of the following formulas:

where R is

R^(b) is

where X is O or NH and R^(c) is

where x is 7-17, and each R^(a), independently, is absent, H, alkyl, orcycloalkyl. In some cases, R^(a) is alkyl or cycloalkyl for no more thantwo occurrences, or R^(a) can be alkyl or cycloalkyl for no more thanone occurrence. In some cases, R^(a) is methyl.

In another aspect, the lipid can have the formula:

wherein X

is —CH₂—, —O—, —S—, —SS—, —CO—, —C(O)O—, —C(O)N(R′)—, —OC(O)N(R′)—,—N(R′)C(O)N(R″)—, —C(O)—N(R′)—N═C(R′″)—; —N(R′)—N═C(R″)—, —O—N═C(R″)—,—C(S)O—, —C(S)N(R′)—, —OC(S) N(R′)—, —N(R′)C(S)N(R″)—,—C(S)—N(R′)—N═C(R′″); —S—N═C(R″); —C(O)S—, —SC(O)N(R′)—, —OC(O)—,—N(R′)C(O)—, —N(R′)C(O)O—, —C(R′″)═N—N(R′)—; —C(R′″)═N—N(R′)—C(O)—,—C(R′″)═N—O—, —OC(S)—, —SC(O)—, —N(R′)C(S)—, —N(R′)C(S)O—, —N(R′)C(O)S—,—C(R′″)═N—N(R′)—C(S)—, —C(R′″)═N—S—, C[═N(R′)]O,

C[═N(R′)]N(R″), —OC[═N(R′)]—, —N(R″)C[═N(R′)]N(R′″)—, —N(R″)C[═N(R′)]—,

Y is N, O or S; and when Y is O or S, then Q₄ and Q₅ are absent.

When Y is N, Q₄ and Q₅ can independently be H, alkyl (e.g., a primary,secondary or tertiary alkyl, such as, for example, Me, Et, isopropyl, ortert-butyl), alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, orheterocyclyl. When Q₄ or Q₅ includes a double or triple bond, the doubleor triple bond can be anywhere in the chain. When there are two or moredouble (or triple) bonds or combination of both then the double (ortriple) bonds can be separated by at least 1 saturated carbon atom. Insome occurrences, two or more multiple bonds can be conjugated. In someoccurrences, two double bonds are tied to the same carbon atom. Doublebonds can be cis, trans, or combination of cis and trans.

L₁, L₂ and L₃ are each independently alkyl (e.g., primary, secondary ortertiary alkyl, such as, for example, Me, Et, isopropyl, or tert-butyl),aryl, aralkyl, alkenyl or alkynyl.

R₁ is a C₆ to C₆₀ group selected from alkyl, alkenyl, alkynyl,heteroalkyl, aralkyl, cycloalkyl, and heterocyclyl. When Q₄ or Q₅includes a double or triple bond, the double or triple bond can beanywhere in the chain. When there are two or more double (or triple)bonds or combination of both then the double (or triple) bonds can beseparated by at least 1 saturated carbon atom. In some occurrences, twoor more multiple bonds can be conjugated. In some occurrences, twodouble bonds are tied to the same carbon atom. Double bonds can be cis,trans, or combination of cis and trans. In some embodiments, R₁ and R₂are independently branched alkyl. In one example branched alkyl include

wherein R₁₀₀ is independently selected from oleyl, linoleyl, steryl,palmityl and the like.

R′, R″, and R′″ are independently H, or a C₁ to C₃₀ group selected fromalkyl, alkenyl, alkynyl, heteroalkyl, aralkyl, cycloalkyl andheterocyclyl. When R′, R″, or R′″ includes a double or triple bond, thedouble or triple bond can be anywhere in the chain. When there are twoor more double (or triple) bonds or combination of both then the double(or triple) bonds can be separated by at least 1 saturated carbon atom.In some occurrences, two or more multiple bonds can be conjugated. Insome occurrences, two double bonds are tied to the same carbon atom.Double bonds can be cis, trans, or combination of cis and trans.

Each of Z₁, Z₂, Z₃, and Z₄, is independently H, F, XR₁, N(Q₆)(Q₇) or—[Y(Q₄,Q₅)—[C(Z₁,Z₂)]_(p)—N(Q₁,Q₂,Q₃), or a C₁ to C₃₀ group selectedfrom alkyl, substituted alkyl, heteroalkyl, aralkyl, cycloalkyl andheterocyclyl. In some occurrence Z₁ is (═O), (═S) or (═NR′) and Z₂ isabsent.

p is 0 to 19. q is 0 to 20. r is 0 to 100.

Additional synthetic techniques for making lipids can be found inprovisional U.S. Patent Application No. 61/333,122, filed May 10, 2010,which is incorporated by reference in its entirety.

TABLE 1 Some exemplary quaternary amine lipids

The lipid including a quaternary amine can be in the form of a salt,i.e. complexed with a counterion. The counterion can be any anion, suchas an organic or inorganic anion. Suitable examples of such anionsinclude tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, andα-glycerophosphate. Inorganic can include chloride, sulfate, nitrate,bicarbonate, and carbonate salts.

Generally, a lipid including a quaternary amine can be prepared from acorresponding lipid that includes a tertiary amine. The tertiary amineis converted to a quaternary amine by, e.g., alkylation with anappropriate alkyl halide. For example, a lipid including a dimethylaminogroup (i.e., a tertiary amine) can be converted to the correspondingtrimethylamino group by reaction with methyl chloride. Methods formaking lipids including tertiary amine groups are described in, forexample, in application no. PCT/US09/63933, filed Nov. 10, 2009, andapplications referred to therein, including No. 61/113,179, filed Nov.10, 2008; No. 61/154,350, filed Feb. 20, 2009; No. 61/171,439, filedApr. 21, 2009; No. 61/185,438, filed Jun. 9, 2009; No. 61/225,898, filedJul. 15, 2009; and No. 61/234,098, filed Aug. 14, 2009; each of thesedocuments is incorporated by reference in its entirety. In general, thelipids described in these applications are suitable for converting tothe corresponding quaternary amine. See, for example, Table 1 ofapplication no. PCT/US09/63933, filed Nov. 10, 2009, at pages 33-50.

In particular embodiments, the lipids are charged lipids. As usedherein, the term “charged lipid” is meant to include those lipids havingone or two fatty acyl or fatty alkyl chains and a quaternary amino headgroup. The quaternary amine carries a permanent positive charge. Thehead group can optionally include a ionizable group, such as a primary,secondary, or tertiary amine that may be protonated at physiological pH.The presence of the quaternary amine can alter the pKa of the ionizablegroup relative to the pKa of the group in a structurally similarcompound that lacks the quaternary amine (e.g., the quaternary amine isreplaced by a tertiary amine) In some embodiments, a charged lipid isreferred to as an “amino lipid.”

Other charged lipids would include those having alternative fatty acidgroups and other quaternary groups, including those in which the alkylsubstituents are different (e.g., N-ethyl-N-methylamino-,N-propyl-N-ethylamino- and the like). For those embodiments in which R₁and R₂ are both long chain alkyl or acyl groups, they can be the same ordifferent. In general, lipids (e.g., a charged lipid) having lesssaturated acyl chains are more easily sized, particularly when thecomplexes are sized below about 0.3 microns, for purposes of filtersterilization. Charged lipids containing unsaturated fatty acids withcarbon chain lengths in the range of C₁₀ to C₂₀ are typical. Otherscaffolds can also be used to separate the amino group (e.g., the aminogroup of the charged lipid) and the fatty acid or fatty alkyl portion ofthe charged lipid. Suitable scaffolds are known to those of skill in theart.

In certain embodiments, charged lipids of the present invention have atleast one protonatable or deprotonatable group, such that the lipid ispositively charged at a pH at or below physiological pH (e.g. pH 7.4),and neutral at a second pH, preferably at or above physiological pH.Such lipids are also referred to as charged lipids. It will, of course,be understood that the addition or removal of protons as a function ofpH is an equilibrium process, and that the reference to a charged or aneutral lipid refers to the nature of the predominant species and doesnot require that all of the lipid be present in the charged or neutralform. Lipids that have more than one protonatable or deprotonatablegroup, or which are zwiterrionic, are not excluded from use in theinvention.

In certain embodiments, protonatable lipids (i.e., charged lipids)according to the invention have a pKa of the protonatable group in therange of about 4 to about 11. Typically, lipids will have a pKa of about4 to about 7, e.g., between about 5 and 7, such as between about 5.5 and6.8, when incorporated into lipid particles. Such lipids will becationic at a lower pH formulation stage, while particles will belargely (though not completely) surface neutralized at physiological pHaround pH 7.4. One of the benefits of a pKa in the range of betweenabout 4 and 7 is that at least some nucleic acid associated with theoutside surface of the particle will lose its electrostatic interactionat physiological pH and be removed by simple dialysis; thus greatlyreducing the particle's susceptibility to clearance. pKa measurements oflipids within lipid particles can be performed, for example, by usingthe fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic acid (TNS),using methods described in Cullis et al., (1986) Chem Phys Lipids 40,127-144.

The compositions described herein can include mixtures of chargedlipids. For example, the compositions (e.g., lipoplexes and/or lipidnanoparticles) can include lipids that have quaternary amines and lipidsthat do not have quaternary amines, but do have a protonatable aminegroup. Suitable lipids for the compositions (both as quaternary ornon-quaternary amines) include those described in WO 2010/054406, WO2010/054405, WO 2010/054401, WO 2010/054384, U.S. Application No.61/309,697, filed Mar. 2, 2010; U.S. Application No. 61/321,829, filedApr. 7, 2010; U.S. Application No. 61/369,530, filed Jul. 30, 2010; U.S.Application No. 61/333,122, filed May 10, 2010; U.S. Application No.61/369,535, filed Jul. 30, 2010; and U.S. Application No. 61/351,146,filed Jun. 3, 2010, each of which is incorporated by reference in itsentirety.

Cryoprotectants and Freeze-Drying

The formulations can include a cryoprotectant. A formulation can besuspended in a buffer containing a cryprotectant at a volume measured toobtain a final desired lipid concentration. The suspension can beagitated to thoroughly mix the cryoprotectant with the lipidnanoparticles. The suspension can be extruded or filtered to selectnanoparticles of a given size. This can result in a final formulation,which can be stored under appropriate conditions until use. Afterstorage of the lipid nanoparticles with a cryoprotectant, the lipidnanoparticles can be used for delivery of nucleic acids to cells withoutincreased cell death or decreased delivery efficiency.

A cryoprotectant can be a compound used to protect the formulation fromdamage due to cold, for example, freezing. A cryoprotectant can includea polyol, e.g., a carbohydrate, for example, sucrose, trehalose, glucoseor a 2-hydroxypropyl-α-cyclodextrin. A sugar alcohol, such as sorbitol,can also be included in a cryoprotectant. A cryprotectant can include aprotein, a peptide or an amino acid. For example, a cryoprotectant caninclude proline or hydroxyl proline. An organic compound, such asglycerol, ethylene glycol, or propylene glycol, can be included in acryoprotectant. In some instances, a cryoprotectant can include apolymer, for example, polyvinylpyrrolidone, polyethylene glycol orgelatin or hydroxyethylcellulose.

A formulation can be mixed and solvent can be removed, which can resultin a residue. The residue can be resuspended in a buffer including acryoprotectant, or the residue can be resuspended in a buffer and then acryoprotectant can be added. The result can be a suspension offormulation in buffer. The formulation can include lipid nanoparticles.The lipid nanoparticles can also include a nucleic acid. The buffer canbe a buffer solution or a buffered media. The pH of the buffer can begreater than 5.0, greater than 6.0, greater than 6.5, greater than 7.0,greater than 7.1, greater than 7.2, greater than 7.3, greater than 7.4,greater than 7.5, greater than 7.6, greater than 7.7, greater than 7.8,greater than 7.9, greater than 8.0 or greater than 9.0. The pH of thebuffer can be less than 9.0, less than 8.0, less than 7.9, less than7.8, less than 7.7, less than 7.6, less than 7.5, less than 7.4, lessthan 7.3, less than 7.2, less than 7.1, less than 7.0, less than 6.5,less than 6.0 or less than 5.0. The buffer can be pH 7.4. The suspensioncan include less than 20%, less than 15%, less than 12%, less than 10%,less than 9%, less than 8%, less than 7%, less than 6%, less than 5% orless than 3% cryoprotectant by volume. The suspension can include morethan 3%, more than 5%, more than 6%, more than 7%, more than 8%, morethan 9%, more than 10% more than 12%, more than 15%, or more than 20%cryoprotectant by volume. In particular, the suspension can include 5%or 10% cryoprotectant by volume.

Once the formulation residue is resuspended, the lipid concentrationwithin the suspension can be greater than 0.25 mg/mL, greater than 0.5mg/mL, greater than 1.0 mg/mL or greater than 1.5 mg/mL. The lipidconcentration within the suspension can be less than 2.0 mg/mL, lessthan 1.5 mg/mL, less than 1.0 mg/mL, less than 0.5 mg/mL, or less than0.25 mg/mL. More specifically, the concentration can be 1.0 mg/mL.

The suspension can be mixed or agitated to distribute the cryoprotectantthroughout the suspension. The mixing or agitation can occur at 4° C.,25° C. or 37° C. The mixing or agitation can occur for greater than 5minutes, greater than 10 minutes, greater than 15 minutes, greater than30 minutes or greater than an hour. Mixing or agitation can occur byshaking, pipetting or stirring.

After agitation, the lipid nanoparticles contained in the suspension canbe selected for size. For example, the nanoparticles can be filtered orextruded. In some cases, the resuspension can be extruded through afilter, for example a polycarbonate filter. The resuspension can also besyringe filtered. In either case, the filter can allow particles lessthan 0.5 μm, less than 0.45 μm, less than 0.4 μm, less than 0.35 μm,less than 0.3 μm, less than 0.25 μm, less than 0.2 μm or less than 0.15μm to pass through. Specifically, a filter can have a pore size of 0.45μm, 0.4 μm, 0.22 pin or 0.2 μm.

Once the lipid nanoparticles have been filtered or extruded, a finalsuspension of the formulation can contain only lipid nanoparticlessmaller than the pore size of the filter. Lipid nanoparticle sizes canbe less than 300 nm, less than 275 nm, less than 250 nm, less than 225nm, less than 200 nm, less than 175 nm, less than 150 nm, less than 125nm or less than 100 nm.

The final suspension of the formulation can be stored at coldtemperatures, for example, less than or equal to 25° C., less than orequal to 4° C., less than or equal to 0° C., than or equal to −20° C. orless than or equal to −80° C., until the formulation is used. The coldformulation can be prepared for use by warming the formulation (e.g., atroom temperature) until the formulation is at room temperature oradequately thawed.

The final suspension of the formulation can also be stored at lowmoisture. For example, the formulation can be dried, lyophilized orfreeze-dried. Freeze-drying can be accomplished by storing theformulation at −80° C. and then lyophilizing the formulation. The lowmoisture formulation can be prepared for use by rehydrating theformulation, for instance, by resuspending the formulation in a liquid.The liquid can be water, a buffer solution or cell culture media.

The formulation can be stored at cold temperatures or low moisture forgreater than 1 hour, greater than 2 hours, greater than 6 hours, greaterthan 12 hours, greater than 24 hours, greater than 2 days, greater than3 days, greater than 4, greater than 5 days, greater than 6 days orgreater than 1 week and still remain an effective transfection reagent.The formulation can be used for transfections after being stored at coldtemperature or at low moisture.

After storage, the formulation can be reconstituted. For example, alyophilized formulation can be reconstituted by warming following byresuspension; or simply by resuspension in a cold or warm liquid (e.g.,water, buffer or media). A formulation stored as a cold or frozensolution can be reconstituted by warming to a desired temperature, e.g.,4° C., room temperature, or 37° C. Reconstitution can also includealtering the formulation. Formulations may be stored with or withoutnucleic acids included; when stored without a nucleic acid present,reconsistitution can include adding a nucleic acid to the formulation.Altering the formulation can also include adding additional or differentlipids to the formulation. The order of various steps of reconstitutionmay be varied.

Apolipoproteins

In one embodiment, the formulations of the invention further comprise anapolipoprotein. As used herein, the term “apolipoprotein” or“lipoprotein” refers to apolipoproteins known to those of skill in theart and variants and fragments thereof and to apolipoprotein agonists,analogues or fragments thereof described below.

Suitable apolipoproteins include, but are not limited to, ApoA-I,ApoA-II, ApoA-IV, ApoA-V and ApoE, and active polymorphic forms,isoforms, variants and mutants as well as fragments or truncated formsthereof. In certain embodiments, the apolipoprotein is a thiolcontaining apolipoprotein. “Thiol containing apolipoprotein” refers toan apolipoprotein, variant, fragment or isoform that contains at leastone cysteine residue. The most common thiol containing apolipoproteinsare ApoA-I Milano (ApoA-I_(M)) and ApoA-I Paris (ApoA-I_(P)) whichcontain one cysteine residue (Jia et al., 2002, Biochem. Biophys. Res.Comm. 297: 206-13; Bielicki and Oda, 2002, Biochemistry 41: 2089-96).ApoA-II, ApoE2 and ApoE3 are also thiol containing apolipoproteins.Isolated ApoE and/or active fragments and polypeptide analogues thereof,including recombinantly produced forms thereof, are described in U.S.Pat. Nos. 5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189;5,168,045; 5,116,739; the disclosures of which are herein incorporatedby reference. ApoE3 is disclosed in Weisgraber, et al., “Human Eapoprotein heterogeneity: cysteine-arginine interchanges in the aminoacid sequence of the apo-E isoforms,” J. Biol. Chem. (1981) 256:9077-9083; and Rall, et al., “Structural basis for receptor bindingheterogeneity of apolipoprotein E from type III hyperlipoproteinemicsubjects,” Proc. Nat. Acad. Sci. (1982) 79: 4696-4700. See also GenBankaccession number K00396.

In certain embodiments, the apolipoprotein can be in its mature form, inits preproapolipoprotein form or in its proapolipoprotein form. Homo-and heterodimers (where feasible) of pro- and mature ApoA-I (Duverger etal., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-1Milano (Kion et al., 2000, Biophys. J. 79:(3)1679-87; Franceschini etal., 1985, J. Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al.,1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol.Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem.259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem.201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem.258(14):8993-9000) can also be utilized within the scope of theinvention.

In certain embodiments, the apolipoprotein can be a fragment, variant orisoform of the apolipoprotein. The term “fragment” refers to anyapolipoprotein having an amino acid sequence shorter than that of anative apolipoprotein and which fragment retains the activity of nativeapolipoprotein, including lipid binding properties. By “variant” ismeant substitutions or alterations in the amino acid sequences of theapolipoprotein, which substitutions or alterations, e.g., additions anddeletions of amino acid residues, do not abolish the activity of nativeapolipoprotein, including lipid binding properties. Thus, a variant cancomprise a protein or peptide having a substantially identical aminoacid sequence to a native apolipoprotein provided herein in which one ormore amino acid residues have been conservatively substituted withchemically similar amino acids. Examples of conservative substitutionsinclude the substitution of at least one hydrophobic residue such asisoleucine, valine, leucine or methionine for another. Likewise, thepresent invention contemplates, for example, the substitution of atleast one hydrophilic residue such as, for example, between arginine andlysine, between glutamine and asparagine, and between glycine and serine(see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The term“isoform” refers to a protein having the same, greater or partialfunction and similar, identical or partial sequence, and may or may notbe the product of the same gene and usually tissue specific (seeWeisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J.Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem.260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon etal., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vase. Biol.18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90;Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov etal., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985,J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem.255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sacre etal., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003, Biophys.Chem. 100(1-3):481-92; Gong et al., 2002, J. Biol. Chem.277(33):29919-26; Ohta et al., 1984, J. Biol. Chem. 259(23):14888-93 andU.S. Pat. No. 6,372,886).

In certain embodiments, the methods and compositions of the presentinvention include the use of a chimeric construction of anapolipoprotein. For example, a chimeric construction of anapolipoprotein can be comprised of an apolipoprotein domain with highlipid binding capacity associated with an apolipoprotein domaincontaining ischemia reperfusion protective properties. A chimericconstruction of an apolipoprotein can be a construction that includesseparate regions within an apolipoprotein (i.e., homologousconstruction) or a chimeric construction can be a construction thatincludes separate regions between different apolipoproteins (i.e.,heterologous constructions). Compositions comprising a chimericconstruction can also include segments that are apolipoprotein variantsor segments designed to have a specific character (e.g., lipid binding,receptor binding, enzymatic, enzyme activating, antioxidant orreduction-oxidation property) (see Weisgraber 1990, J. Lipid Res.31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35;Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al, 1986, J.Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem.259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al.,1998, Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al.,1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, DrugMetab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem.275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71;Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sorenson et al., 1999,Arterioscler. Thromb. Vasc. Biol. 19(9):2214-25; Palgunachari 1996,Arterioscler. Throb. Vasc. Biol. 16(2):328-38: Thurberg et al., J. Biol.Chem. 271(11):6062-70; Dyer 1991, J. Biol. Chem. 266(23):150009-15; Hill1998, J. Biol. Chem. 273(47):30979-84).

Apolipoproteins utilized in the invention also include recombinant,synthetic, semi-synthetic or purified apolipoproteins. Methods forobtaining apolipoproteins or equivalents thereof, utilized by theinvention are well-known in the art. For example, apolipoproteins can beseparated from plasma or natural products by, for example, densitygradient centrifugation or immunoaffinity chromatography, or producedsynthetically, semi-synthetically or using recombinant DNA techniquesknown to those of the art (see, e.g., Mulugeta et al., 1998, J.Chromatogr. 798(1-2): 83-90; Chung et al., 1980, J. Lipid Res.21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29; Persson,et al., 1998, J. Chromatogr. 711:97-109; U.S. Pat. Nos. 5,059,528,5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 andWO 87/02062).

Apolipoproteins utilized in the invention further include apolipoproteinagonists such as peptides and peptide analogues that mimic the activityof ApoA-I, ApoA-I Milano (ApoA-I_(M)), ApoA-I Paris (ApoA-I_(P)),ApoA-IV, and ApoE. For example, the apolipoprotein can be any of thosedescribed in U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166, and5,840,688, the contents of which are incorporated herein by reference intheir entireties.

Apolipoprotein agonist peptides or peptide analogues can be synthesizedor manufactured using any technique for peptide synthesis known in theart including, e.g., the techniques described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166. For example, the peptides may beprepared using the solid-phase synthetic technique initially describedby Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Other peptidesynthesis techniques may be found in Bodanszky et al., PeptideSynthesis, John Wiley & Sons, 2d Ed., (1976) and other referencesreadily available to those skilled in the art. A summary of polypeptidesynthesis techniques can be found in Stuart and Young, Solid PhasePeptide. Synthesis, Pierce Chemical Company, Rockford, Ill., (1984).Peptides may also be synthesized by solution methods as described in TheProteins, Vol. II, 3d Ed., Neurath et. al., Eds., p. 105-237, AcademicPress, New York, N.Y. (1976). Appropriate protective groups for use indifferent peptide syntheses are described in the above-mentioned textsas well as in McOmie, Protective Groups in Organic Chemistry, PlenumPress, New York, N.Y. (1973). The peptides of the present inventionmight also be prepared by chemical or enzymatic cleavage from largerportions of, for example, apolipoprotein A-I.

In certain embodiments, the apolipoprotein can be a mixture ofapolipoproteins. In one embodiment, the apolipoprotein can be ahomogeneous mixture, that is, a single type of apolipoprotein. Inanother embodiment, the apolipoprotein can be a heterogeneous mixture ofapolipoproteins, that is, a mixture of two or more differentapolipoproteins. Embodiments of heterogenous mixtures of apolipoproteinscan comprise, for example, a mixture of an apolipoprotein from an animalsource and an apolipoprotein from a semi-synthetic source. In certainembodiments, a heterogenous mixture can comprise, for example, a mixtureof ApoA-I and ApoA-I Milano. In certain embodiments, a heterogeneousmixture can comprise, for example, a mixture of ApoA-I Milano and ApoA-IParis. Suitable mixtures for use in the methods and compositions of theinvention will be apparent to one of skill in the art.

If the apolipoprotein is obtained from natural sources, it can beobtained from a plant or animal source. If the apolipoprotein isobtained from an animal source, the apolipoprotein can be from anyspecies. In certain embodiments, the apolipoprotien can be obtained froman animal source. In certain embodiments, the apolipoprotein can beobtained from a human source. In preferred embodiments of the invention,the apolipoprotein is derived from the same species as the individual towhich the apolipoprotein is administered.

Transfections

In one aspect, a method for delivering a nucleic acid to a cell caninclude exposing sample cells to a composition containing a chargedlipid. The charged lipid can include the charged lipids describedherein.

A sample cell can include a eukaryotic cell. The eukaryotic cell can bea stem cell, primary cell or a cell in a cell line. The cell line can bea primary cell line, a secondary cell line or an immortalized cell line.Exemplary cell lines can include Chinese hamster ovary (CHO) cells, HeLacells, U20S cells, Caco-2 cells, HT29 cells, NIH3T3 cells, PC12 cells,HepG2 cells, U937 cells, Vero cells, BHK cells, ME-180 cells, A549cells, HEK-293 cells, MCF-7 cells, Jurkat cells, Mdck cells, 3T3 cells,COS-7 cells or GH3 cells. More specifically, the cell line can includeGFP-CHO cells or DG44-C110 cells. The cells can be non-adherentsuspension cells, including, but not limited to, suspension CHO cells,suspension BHK cells, suspension NS0 cells, suspension HeLa cells andsuspension HEK293 cells.

Exposing sample cells to a composition can include contacting the cellswith the composition, adding the composition to the media the cells arecultured in or incubating the cells in a solution containing thecomposition.

In another aspect, a method for delivering a nucleic acid to samplecells can include forming the composition and exposing the sample cellsto the composition.

The composition can be purchased, provided or formed. Charged lipids canbe prepared for use in transfection by forming into liposomes and mixingwith the macromolecules to be introduced into the cell. Macromoleculesthat can be delivered to cells with the transfection reagents can bemacromolecules having at least one negative charge in the molecule. Suchmacromolecules can include, but are not limited to, proteins,polypeptides and nucleic acids, such as RNA and DNA.

Methods of forming liposomes can include, but are not limited to,sonication, extrusion, extended vortexing, reverse evaporation, andhomogenization, which can include microfulidization. Additional methodsof forming liposomes are well known in the art.

Sonication can produce small, unilamellar vesicles (SUV) with diametersin the range of 15-50 nm. Bath sonicators can be instrumentation usedfor preparation of SUV (Avanti Polar Lipids, Inc., 700 Industrial ParkDrive, Alabaster, Ala. 35007). Sonication can be accomplished by placinga test tube containing the suspension in a bath sonicator (or placingthe tip of a sonicator in the test tube) and sonicating for 5-10 minutesabove the gel-liquid crystal transition temperature of the lipid. Meansize and uniformity can be influenced by lipid composition andconcentration, temperature, sonication time, power, volume, andsonicator tuning. Reverse evaporation can be used to form largerliposome vesicles (>1000 nm) known as giant unilamellar vesicles(GUV's).

Another method of forming liposomal compositions can be extrusion. Lipidextrusion can be a technique in which a lipid suspension is forcedthrough a polycarbonate filter with a defined pore size to yieldparticles having a diameter near the pore size of the filter used.Extrusion through filters with pores having an approximately 100 nmdiameter typically can yield large, unilamellar vesicles (LUV) with amean diameter of 120 nm-140 nm. Mean particle size can also depend onlipid composition and can be reproducible from batch to batch.

In some embodimente, the formed liposomes can be approximately 120 nm to800 nm in diameter.

In some embodiments, the composition can further include a nucleic acid.The nucleic acid can be deoxyribonucleic acid (DNA) or ribonucleic acid(RNA). The nucleic acid can include a chemically modified nucleic acid.Chemical modifications can include methylation, acetylation, oxidation,intercalation, thymine dimerization, PEGylation or phosphorylation. Thechemical modifications can include using, for example, aphosphorothioate, methyl phosphonate or phosphoramidate linkage at theinternucleotide phosphodiester bridge. Additionally, the chemicalmodifications can include a modification of the nucleotide base, forexample, 5-propynyl-pyrimidine, or of the sugar, for example, 2′modified sugars.

The nucleic acid can be 10 to 50 nucleotides long. The nucleic acid canbe an oligonucleotide. The oligonucleotide can be 10 to 50 nucleotideslong. The oligonucleotide can be double stranded or single stranded.More particularly, in some embodiments, the nucleic acid can be siRNA ormRNA. The siRNA can be single stranded or double stranded. In otherembodiments, the nucleic acid can be a shRNA, an antisense nucleic acid,a microRNA, an antimicro RNA, an antagomir, a microRNA inhibitor or animmune stimulatory nucleic acid.

In some embodiments, the sample cells can be in suspension. In somecircumstances, the volume of the sample cells in suspension can be atleast 0.050 L, at least 0.1 L, at least 0.5 L, at least 1 L, at least 3L, at least 5 L, at least 10 L, at least 25 L, at least 40 L or morethan 40 L. The suspension can be cultured in a bioreactor, a flask, atube or a tank.

The suspension can be cultured with or without serum.

In some embodiments, a method for delivering a nucleic acid to samplecells can further include culturing untreated control cells that havenot been exposed to the composition. In other words, a culture of cellsis divided into at least two groups of cells including the sample cellsand the untreated control cells. The sample cells are exposed to thecomposition. The untreated control cells are not exposed to thecomposition and provide a negative control to compare sample cellresults against. The untreated control cells can indicate results thatare independent of treatment with the composition.

In some embodiments, a cell density of the sample cells can increaseafter the sample cells have been exposed to the composition. The celldensity can be greater than 0.1×10⁻⁶ cells/mL, greater than 0.5×10⁻⁶cells/mL, greater than 1.0×10⁻⁶ cells/mL, greater than 1.5×10⁻⁶cells/mL, greater than 2.0×10⁻⁶ cells/mL, greater than 2.5×10⁻⁶cells/mL, greater than 3.0×10⁻⁶ cells/mL or greater than 3.5×10⁻⁶cells/mL. The cell density can increase by greater than 0.1×10⁻⁶cells/mL per day, greater than 0.5×10⁻⁶ cells/mL per day, greater than1.0×10⁻⁶ cells/mL per day, greater than 1.5×10⁻⁶ cells/mL per day,greater than 2.0×10⁻⁶ cells/mL per day or greater than 2.5×10⁻⁶ cells/mLper day. In some circumstances, the cell density of the sample cells canincrease exponentially for a period of time after the sample cells havebeen exposed to the composition.

In some embodiments, the cell density of the sample cells can be greaterthan or equal to the cell density of the untreated control cells. Thecell density measurement can be taken one day, two days, three days,four days, five days, six days, 1 week or greater than 1 week after thesample cells have been exposed to the composition.

In some embodiments, the sample cell viability can be greater than 75%,greater than 80%, greater than 85%, greater than 90% or greater than95%. The sample cell viability can be measured one day, two days, threedays, four days, five days or greater than five days after the samplecells have been exposed to the composition.

In some embodiments, a method for delivering a nucleic acid to samplecells can further include measuring a level of a protein in the samplecells and untreated control cells, the protein can be produced from anmRNA that an siRNA delivered into the sample cells is directed against.

The mRNA that an siRNA molecule is directed against can be determined bythe sequence of the siRNA. The siRNA sequence can be complementary tothe sequence of its target mRNA. Therefore, when the siRNA isincorporated into the RISC complex, the RISC complex can bind to themRNA with the sequence complementary to the siRNA and the RISC complexcan cleave the mRNA. This decreases the level of that mRNA in the cell,and consequently, it can decrease the level of protein translated fromthat mRNA.

An siRNA can target RNA other than an mRNA. An siRNA can have a sequencethat is directed against more than one mRNA, thereby affecting thelevels of more than one mRNA and more than one protein.

Measuring a level of a protein can include measuring the quantity of theprotein, measuring an activity of the protein or measuring a downstreameffect of the protein. The downstream effect can include activation ofanother molecule, modification of another molecule or the presence orabsence of another molecule. Measuring a level of the protein can beaccomplished using techniques well known in the art. Techniques formeasuring the quantity of a protein can include an ultravioletabsorption assay, for example 260 nm and 280 nm absorbance reading, aBradford assays, a Lowry, a Biuret assay, a bicinchoninic assay, or aquantitative Western blot. Techniques for measuring the activity of aprotein can include a SDS-Page, a Western blot, a BIAcore assay or anenzyme-linked immunosorbent assay (ELISA).

In some embodiments, the protein level in the sample cells can be lessthan the protein level in the untreated control cells. The protein levelin the sample cells can be less than 40%, less than 50%, less than 60%,less than 70%, less than 75% or less than 80% of the protein level inthe untreated control cells. The protein level in the sample cells andthe protein level in the untreated control cells can be measured oneday, two days, three days, four days, five days, six days, 1 week orgreater than 1 week after the sample cells have been exposed to thecomposition. The protein level in the sample cells and the protein levelin the untreated control cells can be measured one doubling time, twodoubling times, three doubling times, four doubling times, five doublingtimes or greater than five doubling times after the sample cells havebeen exposed to the composition. A doubling time can be the period oftime required for the quantity of cells to double. For example, if ittakes one cell 24 hours to grow and divide to two cells, the doublingtime is 24 hours.

In some embodiments, the cell density of the sample cells can increaseafter the sample cells have been exposed to the composition. The celldensity can be greater than 0.1×10⁻⁶ cells/mL, greater than 0.5×10⁻⁶cells/mL, greater than 1.0×10⁻⁶ cells/mL, greater than 1.5×10⁻⁶cells/mL, greater than 2.0×10⁻⁶ cells/mL, greater than 2.5×10⁻⁶cells/mL, greater than 3.0×10⁻⁶ cells/mL or greater than 3.5×10⁻⁶cells/mL. The cell density can increase by greater than 0.1×10⁻⁶cells/mL per day, greater than 0.5×10⁻⁶ cells/mL per day, greater than1.0×10⁻⁶ cells/mL per day, greater than 1.5×10⁻⁶ cells/mL per day,greater than 2.0×10⁻⁶ cells/mL per day or greater than 2.5×10⁻⁶ cells/mLper day. In some circumstances, the cell density of the sample cells canincrease exponentially for a period of time after the sample cells havebeen exposed to the composition. In some circumstances, the cell densityof the sample cells can increase exponentially for a period of timeafter the sample cells have been exposed to the composition.

In some circumstances, the cell density of the sample cells can begreater than or equal to the cell density of the control cells. The celldensity measurement can be taken one day, two days, three days, fourdays, five days, six days, 1 week or greater than 1 week after thesample cells have been exposed to the composition.

In some embodiments, the sample cell viability can be greater than 75%,greater than 80%, greater than 85%, greater than 90% or greater than95%. The sample cell viability can be measured one day, two days, threedays, four days, five days or greater than five days after the samplecells have been exposed to the composition.

In some embodiments, a method for delivering a nucleic acid to samplecells can further include measuring a level of a protein in the samplecells and the untreated control cells, where the nucleic acid can be ansiRNA and the protein can be produced from an mRNA that the siRNA isdirected against.

The mRNA that an siRNA molecule is directed against can be determined bythe sequence of the siRNA. The siRNA sequence can be complementary tothe sequence of its target mRNA. Therefore, when the siRNA isincorporated into the RISC complex, the RISC complex can bind to themRNA with the sequence complementary to the siRNA and the RISC complexcan cleave the mRNA. This decreases the level of that mRNA in the cell,and consequently, it can decrease the level of protein translated fromthat mRNA.

An siRNA can target RNA other than an mRNA. An siRNA can have a sequencethat is directed against more than one mRNA, thereby affecting thelevels of more than one mRNA and more than one protein.

Measuring a level of a protein can include measuring the quantity of theprotein, measuring an activity of the protein or measuring a downstreameffect of the protein. The downstream effect can include activation ofanother molecule, modification of another molecule or the presence orabsence of another molecule. Measuring a level of the protein can beaccomplished using techniques well known in the art. Techniques formeasuring the quantity of a protein can include an ultravioletabsorption assay, for example 260 nm and 280 nm absorbance reading, aBradford assays, a Lowry, a Biuret assay, a bicinchoninic assay, or aquantitative Western blot. Techniques for measuring the activity of aprotein can include a SDS-Page, a Western blot, a BIAcore assay or anenzyme-linked immunosorbent assay (ELISA).

In some embodiments, the protein level in the sample cells can be lessthan the protein level in the control cells. The protein level in thesample cells can be less than 40%, less than 50%, less than 60%, lessthan 70%, less than 75% or less than 80% of the protein level in thecontrol cells. The protein level in the sample cells and the proteinlevel in the control cells can be measured one day, two days, threedays, four days, five days, six days, 1 week or greater than 1 weekafter the sample cells have been exposed to the composition. The proteinlevel in the sample cells and the protein level in the control cells canbe measured one doubling time, two doubling times, three doubling times,four doubling times, five doubling times or greater than five doublingtimes after the sample cells have been exposed to the composition. Adoubling time can be the period of time required for the quantity ofcells to double. For example, if it takes one cell 24 hours to grow anddivide to two cells, the doubling time is 24 hours.

The transfection methods can be applied to in vitro and in vivotransfection of cells, particularly to transfection of eukaryotic cellsincluding animal cells. The methods can be used to generate transfectedcells which express useful gene products. The methods can also beemployed as a step in the production of transgenic animals. The methodsare useful as a step in any therapeutic method requiring introducing ofnucleic acids into cells. In particular, these methods are useful incancer treatment, in in vivo and ex vivo gene therapy, and in diagnosticmethods. The transfection compositions can be employed as researchreagents in any transfection of cells done for research purposes.Nucleic acids that can be transfected by the methods of include DNA andRNA from any source comprising natural bases or non-natural bases, andinclude those encoding and capable of expressing therapeutic orotherwise useful proteins in cells, those which inhibit undesiredexpression of nucleic acids in cells, those which inhibit undesiredenzymatic activity or activate desired enzymes, those which catalyzereactions (Ribozymes), and those which function in diagnostic assays.

The reagents and methods provided herein can are also readily adapted tointroduce biologically active anionic macromolecules other than nucleicacids including, among others, polyamines, polyamine acids,polypeptides, proteins, biotin, and polysaccharides into cells. Othermaterials useful, for example as therapeutic agents, diagnosticmaterials and research reagents, can be complexed by the polychargedlipid aggregates and delivered into cells by the methods of thisinvention.

The methods and materials are useful in the development and practice ofcell based assays and in the screening of libraries of molecules by cellbased assays. In such assays, one or more cells are contacted with atest compound after the macromolecule, particularly an expressionvector, is introduced into the one or more cells. Preferably, the one ormore cells are contacted with the test compound for a selected time, forexample within 5 days, after the macromolecule is introduced into theone or more cells.

Lipid Particles

The present invention also provides lipid particles comprising one ormore of the charged lipids described above. A complex of nucleic acidand lipid particles can be referred to as an association complex. Anassociation complex of nucleic acid and lipid particle may be aliposome, a nanoparticle, an ion pair, a lipoplex, or a combinationthereof. Lipoplexes are composed of charged lipid bilayers sandwichedbetween DNA layers, as described, e.g., in Feigner, Scientific American.Lipid particles include, but are not limited to, liposomes. As usedherein, a liposome is a structure having lipid-containing membranesenclosing an aqueous interior. Liposomes may have one or more lipidmembranes. The invention contemplates both single-layered liposomes,which are referred to as unilamellar, and multi-layered liposomes, whichare referred to as multilamellar.

The lipid particles of the present invention may further comprise one ormore additional lipids and/or other components such as cholesterol.Other lipids may be included in the liposome compositions of the presentinvention for a variety of purposes, such as to prevent lipid oxidationor to attach ligands onto the liposome surface. Any of a number oflipids may be present in liposomes of the present invention, includingamphipathic, neutral, cationic, and anionic lipids. Such lipids can beused alone or in combination. Specific examples of additional lipidcomponents that may be present are described below.

Additional components that may be present in a lipid particle of thepresent invention include bilayer stabilizing components such aspolyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides,proteins, detergents, lipid-derivatives, such as PEG coupled tophosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat.No. 5,885,613).

In particular embodiments, the lipid particles include one or more of asecond amino lipid or charged lipid, a neutral lipid, and a sterol.

Neutral lipids, when present in the lipid particle, can be any of anumber of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH. Such lipids include, for examplephosphocholines (PC), phosphatidylethanolamines (PE),phosphatidylserines (PS), cardiolipins, diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide, sphingomyelin,dihydrosphingomyelin, cephalin, and cerebrosides. The selection ofneutral lipids for use in the particles described herein is generallyguided by consideration of, e.g., liposome size and stability of theliposomes in the bloodstream. Preferably, the neutral lipid component isa lipid having two acyl groups, (i.e., diacylphosphatidylcholine anddiacylphosphatidylethanolamine). Lipids having a variety of acyl chaingroups of varying chain length and degree of saturation are available ormay be isolated or synthesized by well-known techniques. In one group ofembodiments, lipids containing saturated fatty acids with carbon chainlengths in the range of C₁₀ to C₂₀ are preferred. In another group ofembodiments, lipids with mono or diunsaturated fatty acids with carbonchain lengths in the range of C₁₀ to C₂₀ are used. Additionally, lipidshaving mixtures of saturated and unsaturated fatty acid chains can beused, Preferably, the neutral lipids used in the present invention areDOPE, DSPC, POPC, DPPC or any related phosphatidylcholine. The neutrallipids useful in the present invention may also be composed ofsphingomyelin, dihydrosphingomyeline, or phospholipids with other headgroups, such as serine and inositol.

The sterol component of the lipid mixture, when present, can be any ofthose sterols conventionally used in the field of liposome, lipidvesicle or lipid particle preparation. A preferred sterol ischolesterol.

Other protonatable lipids, which carry a net positive charge at aboutphysiological pH, in addition to those specifically described above, mayalso be included in lipid particles of the present invention. Suchprotonatable lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations oflipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE,available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE,available from GIBCO/BRL).

Anionic lipids suitable for use in lipid particles include, but are notlimited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine,N-succinyl phosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionicmodifying groups joined to neutral lipids.

In numerous embodiments, amphipathic lipids are included in lipidparticles of the present invention. “Amphipathic lipids” refer to anysuitable material, wherein the hydrophobic portion of the lipid materialorients into a hydrophobic phase, while the hydrophilic portion orientstoward the aqueous phase. Such compounds include, but are not limitedto, phospholipids, aminolipids, and sphingolipids. Representativephospholipids include sphingomyelin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatdylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Otherphosphorus-lacking compounds, such as sphingolipids, glycosphingolipidfamilies, diacylglycerols, and β-acyloxyacids, can also be used.Additionally, such amphipathic lipids can be readily mixed with otherlipids, such as triglycerides and sterols.

In some cases, the lipid particle can include a lipid selected to reduceaggregation of lipid particles during formation, which may result fromsteric stabilization of particles which prevents charge-inducedaggregation during formation.

Examples of lipids that reduce aggregation of particles during formationinclude polyethylene glycol (PEG)-modified lipids, monosialogangliosideGm1, and polyamide oligomers (“PAO”) such as (described in U.S. Pat. No.6,320,017). Other compounds with uncharged, hydrophilic, steric-barriermoieties, which prevent aggregation during formulation, like PEG, Gml orATTA, can also be coupled to lipids for use as in the methods andcompositions of the invention. ATTA-lipids are described, e.g., in U.S.Pat. No. 6,320,017, and PEG-lipid conjugates are described, e.g., inU.S. Pat. Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, theconcentration of the lipid component selected to reduce aggregation isabout 1 to 15% (by mole percent of lipids).

Specific examples of PEG-modified lipids (or lipid-polyoxyethyleneconjugates) that are useful in the present invention can have a varietyof “anchoring” lipid portions to secure the PEG portion to the surfaceof the lipid vesicle. Examples of suitable PEG-modified lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which aredescribed in co-pending U.S. Ser. No. 08/486,214, incorporated herein byreference, PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modifieddiacylglycerols and dialkylglycerols.

In embodiments where a sterically-large moiety such as PEG or ATTA areconjugated to a lipid anchor, the selection of the lipid anchor dependson what type of association the conjugate is to have with the lipidparticle. It is well known that mPEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remainassociated with a liposome until the particle is cleared from thecirculation, possibly a matter of days. Other conjugates, such asPEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidlyexchanges out of the formulation upon exposure to serum, with a T_(1/2)less than 60 mins. in some assays. As illustrated in U.S. patentapplication Ser. No. 08/486,214, at least three characteristicsinfluence the rate of exchange: length of acyl chain, saturation of acylchain, and size of the steric-barrier head group. Compounds havingsuitable variations of these features may be useful for the invention.For some therapeutic applications it may be preferable for thePEG-modified lipid to be rapidly lost from the nucleic acid-lipidparticle in vivo and hence the PEG-modified lipid will possessrelatively short lipid anchors. In other therapeutic applications it maybe preferable for the nucleic acid-lipid particle to exhibit a longerplasma circulation lifetime and hence the PEG-modified lipid willpossess relatively longer lipid anchors.

It should be noted that aggregation preventing compounds do notnecessarily require lipid conjugation to function properly. Free PEG orfree ATTA in solution may be sufficient to prevent aggregation. If theparticles are stable after formulation, the PEG or ATTA can be dialyzedaway before administration to a subject.

Also suitable for inclusion in the lipid particles of the presentinvention are programmable fusion lipids. Such lipid particles havelittle tendency to fuse with cell membranes and deliver their payloaduntil a given signal event occurs. This allows the lipid particle todistribute more evenly after injection into an organism or disease sitebefore it starts fusing with cells. The signal event can be, forexample, a change in pH, temperature, ionic environment, or time. In thelatter case, a fusion delaying or “cloaking” component, such as anATTA-lipid conjugate or a PEG-lipid conjugate, can simply exchange outof the lipid particle membrane over time. By the time the lipid particleis suitably distributed in the body, it has lost sufficient cloakingagent so as to be fusogenic. With other signal events, it is desirableto choose a signal that is associated with the disease site or targetcell, such as increased temperature at a site of inflammation.

In certain embodiments, it is desirable to target the lipid particles ofthis invention using targeting moieties that are specific to a cell typeor tissue. Targeting of lipid particles using a variety of targetingmoieties, such as ligands, cell surface receptors, glycoproteins,vitamins (e.g., riboflavin) and monoclonal antibodies, has beenpreviously described (see, e.g., U.S. Pat. Nos. 4,957,773 and4,603,044). The targeting moieties can comprise the entire protein orfragments thereof. Targeting mechanisms generally require that thetargeting agents be positioned on the surface of the lipid particle insuch a manner that the target moiety is available for interaction withthe target, for example, a cell surface receptor. A variety of differenttargeting agents and methods are known and available in the art,including those described, e.g., in Sapra, P. and Allen, T M, Prog.Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res.12:1-3, (2002).

The use of lipid particles, i.e., liposomes, with a surface coating ofhydrophilic polymer chains, such as polyethylene glycol (PEG) chains,for targeting has been proposed (Allen, et al., Biochimica et BiophysicaActa 1237: 99-108 (1995); DeFrees, et al., Journal of the AmericanChemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica etBiophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal ofLiposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353:71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic andMartin, Eds) CRC Press, Boca Raton Fla. (1995). In one approach, aligand, such as an antibody, for targeting the lipid particle is linkedto the polar head group of lipids forming the lipid particle. In anotherapproach, the targeting ligand is attached to the distal ends of the PEGchains forming the hydrophilic polymer coating (Klibanov, et al.,Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBSLetters 388: 115-118 (1996)).

Standard methods for coupling the target agents can be used. Forexample, phosphatidylethanolamine, which can be activated for attachmentof target agents, or derivatized lipophilic compounds, such aslipid-derivatized bleomycin, can be used. Antibody-targeted liposomescan be constructed using, for instance, liposomes that incorporateprotein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990)and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990).Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference.Examples of targeting moieties can also include other proteins, specificto cellular components, including antigens associated with neoplasms ortumors. Proteins used as targeting moieties can be attached to theliposomes via covalent bonds (see, Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

In one exemplary embodiment, the lipid particle comprises a mixture of acharged lipid of the present invention, one or more different neutrallipids, and a sterol (e.g., cholesterol). In certain embodiments, thelipid mixture consists of or consists essentially of a charged lipid asdescribed herein, a neutral lipid, and cholesterol. In further preferredembodiments, the lipid particle consists of or consists essentially ofthe above lipid mixture in molar ratios of about 50-90% charged lipid,0-50% neutral lipid, and 0-10% cholesterol. In certain embodiments, thelipid particle can further include a PEG-modified lipid (e.g., a PEG-DMGor PEG-DMA).

In one embodiment, the lipid particle consists of a charged lipid (e.g.,a quaternary nitrogen containing lipid) and a protonatable lipid, aneutral lipid or a steroid, or a combination thereof. The particles canbe formulated with a nucleic acid therapeutic agent so as to attain adesired N/P ratio. The N/P ratio is the ratio of number of molarequivalent of cationic nitrogen (N) atoms present in the lipid particleto the number of molar equivalent of anionic phosphate (P) of thenucleic acid backbone. For example, the N/P ratio can be in the range ofabout 1 to about 50. In one example, the range is about 1 to about 20,about 1 to about 10, about 1 to about 5.

In particular embodiments, the lipid particle consists of or consistsessentially of a charged lipid, DOPE, and cholesterol. In particularembodiments, the particle includes lipids in the following molepercentages: charged lipid, 45-63 mol %; DOPE, 35-55 mol %; andcholesterol, 0-10 mol %. The particles can be formulated with a nucleicacid therapeutic agent so as to attain a desired N/P ratio. The NIPratio is the ratio of number of moles cationic nitrogen (N) atoms (i.e.,charged lipids) to the number of molar equivalents of anionic phosphate(P) backbone groups of the nucleic acid. For example, the N to P ratiocan be in the range of about 5:1 to about 1:1. In certain embodiments,the charged lipid is chosen from compound 205, 201, or 204 (see Scheme 1below).

In another group of embodiments, the neutral lipid, DOPE, in thesecompositions is replaced with POPC, DPPC, DPSC or SM.

Therapeutic Agent-Lipid Particle Compositions and Formulations

The present invention includes compositions comprising a lipid particleof the present invention and an active agent, wherein the active agentis associated with the lipid particle. In particular embodiments, theactive agent is a therapeutic agent. In particular embodiments, theactive agent is encapsulated within an aqueous interior of the lipidparticle. In other embodiments, the active agent is present within oneor more lipid layers of the lipid particle. In other embodiments, theactive agent is bound to the exterior or interior lipid surface of alipid particle.

“Fully encapsulated” as used herein indicates that the nucleic acid inthe particles is not significantly degraded after exposure to serum or anuclease assay that would significantly degrade free nucleic acids. In afully encapsulated system, preferably less than 25% of particle nucleicacid is degraded in a treatment that would normally degrade 100% of freenucleic acid, more preferably less than 10% and most preferably lessthan 5% of the particle nucleic acid is degraded. Alternatively, fullencapsulation may be determined by an Oligreen® assay. Oligreen® is anultra-sensitive fluorescent nucleic acid stain for quantitatingoligonucleotides and single-stranded DNA in solution (available fromInvitrogen Corporation, Carlsbad, Calif.). Fully encapsulated alsosuggests that the particles are serum stable, that is, that they do notrapidly decompose into their component parts upon in vivoadministration.

Active agents, as used herein, include any molecule or compound capableof exerting a desired effect on a cell, tissue, organ, or subject. Sucheffects may be biological, physiological, or cosmetic, for example.Active agents may be any type of molecule or compound, including e.g.,nucleic acids, peptides and polypeptides, including, e.g., antibodies,such as, e.g., polyclonal antibodies, monoclonal antibodies, antibodyfragments; humanized antibodies, recombinant antibodies, recombinanthuman antibodies, and Primatized™ antibodies, cytokines, growth factors,apoptotic factors, differentiation-inducing factors, cell surfacereceptors and their ligands; hormones; and small molecules, includingsmall organic molecules or compounds.

In one embodiment, the active agent is a therapeutic agent, or a salt orderivative thereof. therapeutic agent derivatives may be therapeuticallyactive themselves or they may be prodrugs, which become active uponfurther modification. Thus, in one embodiment, a therapeutic agentderivative retains some or all of the therapeutic activity as comparedto the unmodified agent, while in another embodiment, a therapeuticagent derivative lacks therapeutic activity.

In various embodiments, therapeutic agents include any therapeuticallyeffective agent or drug, such as anti-inflammatory compounds,anti-depressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs, e.g.,anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.

In certain embodiments, the therapeutic agent is an oncology drug, whichmay also be referred to as an anti-tumor drug, an anti-cancer drug, atumor drug, an antineoplastic agent, or the like. Examples of oncologydrugs that may be used according to the invention include, but are notlimited to, adriamycin, alkeran, allopurinol, altretamine, amifostine,anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU,bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda),carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin,cladribine, cyclosporin A, cytarabine, cytosine arabinoside,daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane,dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine,etoposide phosphate, etoposide and VP-16, exemestane, FK506,fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar),gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea,idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan(Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide,levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna,methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone,nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin,porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen,taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin),toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine,vincristine, VP16, and vinorelbine. Other examples of oncology drugsthat may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors and camptothecins.

Nucleic Acid-Lipid Particles

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle. In particular embodiments, the nucleic acid is fullyencapsulated in the lipid particle. As used herein, the term “nucleicacid” is meant to include any oligonucleotide or polynucleotide.Fragments containing up to 50 nucleotides are generally termedoligonucleotides, and longer fragments are called polynucleotides. Inparticular embodiments, oligonucletoides of the present invention are15-50 nucleotides in length.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also includes polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

The nucleic acid that is present in a lipid-nucleic acid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA include structural genes, genes including controland termination regions, and self-replicating systems such as viral orplasmid DNA. Examples of double-stranded RNA include siRNA and other RNAinterference reagents. Single-stranded nucleic acids include, e.g.,antisense oligonucleotides, ribozymes, microRNA, siRNA, antimicroRNA,antagomirs, microRNA inhibitor, supermirs, and triplex-formingoligonucleotides. The nucleic acid that is present in a lipid-nucleicacid particle of this invention may include one or more of theoligonucleotide modifications described below.

Nucleic acids of the present invention may be of various lengths,generally dependent upon the particular form of nucleic acid. Forexample, in particular embodiments, plasmids or genes may be from about1,000 to 100,000 nucleotide residues in length. In particularembodiments, oligonucleotides may range from about 10 to 100 nucleotidesin length. In various related embodiments, oligonucleotides,single-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 50 nucleotides, from about 20 o about 50nucleotides, from about 15 to about 30 nucleotides, from about 20 toabout 30 nucleotides in length.

In particular embodiments, the oligonucleotide (or a strand thereof) ofthe present invention specifically hybridizes to or is complementary toa target polynucleotide. “Specifically hybridizable” and “complementary”are terms which are used to indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility or expression therefrom, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or, in the case of invitro assays, under conditions in which the assays are conducted. Thus,in other embodiments, this oligonucleotide includes 1, 2, or 3 basesubstitutions, e.g. mismatches, as compared to the region of a gene ormRNA sequence that it is targeting or to which it specificallyhybridizes.

RNA Interference Nucleic Acids

In particular embodiments, nucleic acid-lipid particles of the presentinvention are associated with RNA interference (RNAi) molecules. RNAinterference methods using RNAi molecules may be used to disrupt theexpression of a gene or polynucleotide of interest. Small interferingRNA (siRNA) has essentially replaced antisense ODN and ribozymes as thenext generation of targeted oligonucleotide drugs under development.

SiRNAs are RNA duplexes normally 16-30 nucleotides long that canassociate with a cytoplasmic multi-protein complex known as RNAi-inducedsilencing complex (RISC). RISC loaded with siRNA mediates thedegradation of homologous mRNA transcripts, therefore siRNA can bedesigned to knock down protein expression with high specificity. Unlikeother antisense technologies, siRNA function through a natural mechanismevolved to control gene expression through non-coding RNA. This isgenerally considered to be the reason why their activity is more potentin vitro and in vivo than either antisense ODN or ribozymes. A varietyof RNAi reagents, including siRNAs targeting clinically relevanttargets, are currently under pharmaceutical development, as described,e.g., in de Fougerolles, A. et al., Nature Reviews 6:443-453 (2007).

While the first described RNAi molecules were RNA:RNA hybrids comprisingboth an RNA sense and an RNA antisense strand, it has now beendemonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNAantisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi(Lamberton, J. S. and Christian, A. T., (2003) Molecular Biotechnology24:111-119). Thus, the invention includes the use of RNAi moleculescomprising any of these different types of double-stranded molecules. Inaddition, it is understood that RNAi molecules may be used andintroduced to cells in a variety of forms. Accordingly, as used herein,RNAi molecules encompasses any and all molecules capable of inducing anRNAi response in cells, including, but not limited to, double-strandedoligonucleotides comprising two separate strands, i.e. a sense strandand an antisense strand, e.g., small interfering RNA (siRNA);double-stranded oligonucleotide comprising two separate strands that arelinked together by non-nucleotidyl linker; oligonucleotides comprising ahairpin loop of complementary sequences, which forms a double-strandedregion, e.g., shRNAi molecules, and expression vectors that express oneor more polynucleotides capable of forming a double-strandedpolynucleotide alone or in combination with another polynucleotide.

A “single strand siRNA compound” as used herein, is an siRNA compoundwhich is made up of a single molecule. It may include a duplexed region,formed by intra-strand pairing, e.g., it may be, or include, a hairpinor pan-handle structure. Single strand siRNA compounds may be antisensewith regard to the target molecule

A single strand siRNA compound may be sufficiently long that it canenter the RISC and participate in RISC mediated cleavage of a targetmRNA. A single strand siRNA compound is at least 14, and in otherembodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides inlength. In certain embodiments, it is less than 200, 100, or 60nucleotides in length.

Hairpin siRNA compounds will have a duplex region equal to or at least17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplexregion will may be equal to or less than 200, 100, or 50, in length. Incertain embodiments, ranges for the duplex region are 15-30, 17 to 23,19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin may havea single strand overhang or terminal unpaired region. In certainembodiments, the overhangs are 2-3 nucleotides in length. In someembodiments, the overhang is at the sense side of the hairpin and insome embodiments on the antisense side of the hairpin.

A “double stranded siRNA compound” as used herein, is an siRNA compoundwhich includes more than one, and in some cases two, strands in whichinterchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded siRNA compound may be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It may be equal to or less than 200, 100, or 50, nucleotides inlength. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength. As used herein, term “antisense strand” means the strand of ansiRNA compound that is sufficiently complementary to a target molecule,e.g. a target RNA.

The sense strand of a double stranded siRNA compound may be equal to orat least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length.It may be equal to or less than 200, 100, or 50, nucleotides in length.Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The double strand portion of a double stranded siRNA compound may beequal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29,40, or 60 nucleotide pairs in length. It may be equal to or less than200, 100, or 50, nucleotides pairs in length. Ranges may be 15-30, 17 to23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the siRNA compound is sufficiently large that itcan be cleaved by an endogenous molecule, e.g., by Dicer, to producesmaller siRNA compounds, e.g., siRNAs agents

The sense and antisense strands may be chosen such that thedouble-stranded siRNA compound includes a single strand or unpairedregion at one or both ends of the molecule. Thus, a double-strandedsiRNA compound may contain sense and antisense strands, paired tocontain an overhang, e.g., one or two 5′ or 3′ overhangs, or a 3′overhang of 1-3 nucleotides. The overhangs can be the result of onestrand being longer than the other, or the result of two strands of thesame length being staggered. Some embodiments will have at least one 3′overhang. In one embodiment, both ends of an siRNA molecule will have a3′ overhang. In some embodiments, the overhang is 2 nucleotides.

In certain embodiments, the length for the duplexed region is between 15and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe ssiRNA compound range discussed above. ssiRNA compounds can resemblein length and structure the natural Dicer processed products from longdsiRNAs. Embodiments in which the two strands of the ssiRNA compound arelinked, e.g., covalently linked are also included. Hairpin, or othersingle strand structures which provide the required double strandedregion, and a 3′ overhang are also within the invention.

The siRNA compounds described herein, including double-stranded siRNAcompounds and single-stranded siRNA compounds can mediate silencing of atarget RNA, e.g., mRNA, e.g. a transcript of a gene that encodes aprotein. For convenience, such mRNA is also referred to herein as mRNAto be silenced. Such a gene is also referred to as a target gene. Ingeneral, the RNA to be silenced is an endogenous gene or a pathogengene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs,can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an ssiRNA compound of 21 to23 nucleotides.

In one embodiment, an siRNA compound is “sufficiently complementary” toa target RNA, e.g., a target mRNA, such that the siRNA compound silencesproduction of protein encoded by the target mRNA. In another embodiment,the siRNA compound is “exactly complementary” to a target RNA, e.g., thetarget RNA and the siRNA compound anneal, for example to form a hybridmade exclusively of Watson-Crick base pairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can includean internal region (e.g., of at least 10 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in certain embodiments, thesiRNA compound specifically discriminates a single-nucleotidedifference. In this case, the siRNA compound only mediates RNAi if exactcomplementary is found in the region (e.g., within 7 nucleotides of) thesingle-nucleotide difference.

MicroRNAs

Micro RNAs (miRNAs) are a highly conserved class of small RNA moleculesthat are transcribed from DNA in the genomes of plants and animals, butare not translated into protein. Processed miRNAs are single stranded˜17-25 nucleotide (nt) RNA molecules that become incorporated into theRNA-induced silencing complex (RISC) and have been identified as keyregulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

The number of miRNA sequences identified to date is large and growing,illustrative examples of which can be found, for example, in: “miRBase:microRNA sequences, targets and gene nomenclature” Griffiths-Jones S,Grocock RJ, van Dongen S, Bateman A, Enright AJ. NAR, 2006, 34, DatabaseIssue, D140-D144; “The microRNA Registry” Griffiths-Jones S, NAR, 2004,32, Database Issue, D109-D111; and also athttp://microrna.sanger.ac.uk/sequences/.

Antisense Oligonucleotides

In one embodiment, a nucleic acid is an antisense oligonucleotidedirected to a target polynucleotide. The term “antisenseoligonucleotide” or simply “antisense” is meant to includeoligonucleotides that are complementary to a targeted polynucleotidesequence. Antisense oligonucleotides are single strands of DNA or RNAthat are complementary to a chosen sequence, e.g. a target gene mRNA.Antisense oligonucleotides are thought to inhibit gene expression bybinding to a complementary mRNA. Binding to the target mRNA can lead toinhibition of gene expression by through making the either by preventingtranslation of complementary mRNA strands by binding to it or by leadingto degradation of the target mRNA Antisense DNA can be used to target aspecific, complementary (coding or non-coding) RNA. If binding takesplaces this DNA/RNA hybrid can be degraded by the enzyme RNase H. Inparticular embodiment, antisense oligonucleotides contain from about 10to about 50 nucleotides, more preferably about 15 to about 30nucleotides. The term also encompasses antisense oligonucleotides thatmay not be exactly complementary to the desired target gene. Thus, theinvention can be utilized in instances where non-targetspecific-activities are found with antisense, or where an antisensesequence containing one or more mismatches with the target sequence isthe most preferred for a particular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examplesof antisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed, CancerCommun. 1989; 1(4):225-32; Pens et al., Brain Res Mol Brain Res. 1998Jun. 15; 57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573;U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Furthermore,antisense constructs have also been described that inhibit and can beused to treat a variety of abnormal cellular proliferations, e.g. cancer(U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end. Antagomirs may be usedto efficiently silence endogenous miRNAs by forming duplexes comprisingthe antagomir and endogenous miRNA, thereby preventing miRNA-inducedgene silencing. An example of antagomir-mediated miRNA silencing is thesilencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438:685-689, which is expressly incorporated by reference herein in itsentirety. Antagomir RNAs may be synthesized using standard solid phaseoligonucleotide synthesis protocols. See U.S. patent application Ser.Nos. 11/502,158 and 11/657,341 (the disclosure of each of which areincorporated herein by reference).

An antagomir can include ligand-conjugated monomer subunits and monomersfor oligonucleotide synthesis. Exemplary monomers are described in U.S.application Ser. No. 10/916,185, filed on Aug. 10, 2004. An antagomircan have a ZXY structure, such as is described in PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004. An antagomir can be complexedwith an amphipathic moiety. Exemplary amphipathic moieties for use witholigonucleotide agents are described in PCT Application No.PCT/US2004/07070, filed on Mar. 8, 2004.

Aptamers

Aptamers are nucleic acid or peptide molecules that bind to a particularmolecule of interest with high affinity and specificity (Tuerk and Gold,Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).DNA or RNA aptamers have been successfully produced which bind manydifferent entities from large proteins to small organic molecules. SeeEaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin.Struct. Biol. 9:324-9 (1999), and Hermann and Patel, Science 287:820-5(2000). Aptamers may be RNA or DNA based, and may include a riboswitch.A riboswitch is a part of an mRNA molecule that can directly bind asmall target molecule, and whose binding of the target affects thegene's activity. Thus, an mRNA that contains a riboswitch is directlyinvolved in regulating its own activity, depending on the presence orabsence of its target molecule. Generally, aptamers are engineeredthrough repeated rounds of in vitro selection or equivalently, SELEX(systematic evolution of ligands by exponential enrichment) to bind tovarious molecular targets such as small molecules, proteins, nucleicacids, and even cells, tissues and organisms. The aptamer may beprepared by any known method, including synthetic, recombinant, andpurification methods, and may be used alone or in combination with otheraptamers specific for the same target. Further, as described more fullyherein, the term “aptamer” specifically includes “secondary aptamers”containing a consensus sequence derived from comparing two or more knownaptamers to a given target.

Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA moleculescomplexes having specific catalytic domains that possess endonucleaseactivity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December;84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24; 49(2):211-20).For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., Cell. 1981 December; 27(3 Pt 2):487-96; Michel and Westhof, JMol. Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurek and Shub, Nature.1992 May 14; 357(6374):173-6). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif, forexample. Specific examples of hammerhead motifs are described by Rossiet al. Nucleic Acids Res. 1992 Sep. 11; 20(17):4559-65. Examples ofhairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No.EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan. 25;18(2):299-304 and U.S. Pat. No. 5,631,359. An example of the hepatitis □virus motif is described by Perrotta and Been, Biochemistry. 1992 Dec.1; 31(47):11843-52; an example of the RNaseP motif is described byGuerrier-Takada et al., Cell. 1983 December; 35(3 Pt 2):849-57;Neurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, Cell. 1990 May 18; 61(4):685-96; Saville and Collins, Proc NatlAcad Sci USA. 1991 Oct. 1; 88(19):8826-30; Collins and Olive,Biochemistry. 1993 Mar. 23; 32(11):2795-9); and an example of the GroupI intron is described in U.S. Pat. No. 4,987,071. Importantcharacteristics of enzymatic nucleic acid molecules used according tothe invention are that they have a specific substrate binding site whichis complementary to one or more of the target gene DNA or RNA regions,and that they have nucleotide sequences within or surrounding thatsubstrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in Int.Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO94/02595, each specifically incorporated herein by reference, andsynthesized to be tested in vitro and in vivo, as described therein.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem H bases toshorten RNA synthesis times and reduce chemical requirements.

Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles of the present inventionmay be immunostimulatory, including immunostimulatory oligonucleotides(ISS; single- or double-stranded) capable of inducing an immune responsewhen administered to a subject, which may be a mammal or other patient.ISS include, e.g., certain palindromes leading to hairpin secondarystructures (see Yamamoto S., et al. (1992) J. Immunol. 148: 4072-4076),or CpG motifs, as well as other known ISS features (such as multi-Gdomains, see WO 96/11266).

The immune response may be an innate or an adaptive immune response. Theimmune system is divided into a more innate immune system, and acquiredadaptive immune system of vertebrates, the latter of which is furtherdivided into humoral cellular components. In particular embodiments, theimmune response may be mucosal.

In particular embodiments, an immunostimulatory nucleic acid is onlyimmunostimulatory when administered in combination with a lipidparticle, and is not immunostimulatory when administered in its “freeform.” According to the present invention, such an oligonucleotide isconsidered to be immunostimulatory.

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target polynucleotide in order to provoke animmune response. Thus, certain immunostimulatory nucleic acids maycomprise a seuqence correspondign to a region of a naturally occurringgene or mRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein said CpG dinucleotide is methylated. In a specific embodiment, thenucleic acid comprises the sequence 5′ TAACGTTGAGGGGCAT 3′. In analternative embodiment, the nucleic acid comprises at least two CpGdinucleotides, wherein at least one cytosine in the CpG dinucleotides ismethylated. In a further embodiment, each cytosine in the CpGdinucleotides present in the sequence is methylated. In anotherembodiment, the nucleic acid comprises a plurality of CpG dinucleotides,wherein at least one of said CpG dinucleotides comprises a methylatedcytosine.

In one specific embodiment, the nucleic acid comprises the sequence5′TTCCATGACGTTCCTGACGT 3′. In another specific embodiment, the nucleicacid sequence comprises the sequence 5′ TCCATGACGTTCCTGACGT 3′, whereinthe two cytosines indicated in bold are methylated. In particularembodiments, the ODN is selected from a group of ODNs consisting of ODN#1, ODN #2, ODN #3, ODN #4, ODN #5, ODN #6, ODN #7, ODN #8, and ODN #9,as shown below.

TABLE 3 Exemplary Immunostimulatory Oligonucleotides (ODNs) SEQ ODN NAMEID ODN SEQUENCE (5′-3′). ODN 1 5′-TAACGTTGAGGGGCAT-3 human c-myc* ODN 1m 5′-TAAZGTTGAGGGGCAT-3 ODN 2 5′-TCCATGACGTTCCTGACGTT-3 * ODN 2m5′-TCCATGAZGTTCCTGAZGTT-3 ODN 3 5′-TAAGCATACGGGGTGT-3 ODN 5 5′-AACGTT-3ODN 6 5′- GATGCTGTGTCGGGGTCTCCGGGC- 3′ ODN 75′-TCGTCGTTTTGTCGTTTTGTCGTT- 3′ ODN 7m 5′-TZGTZGTTTTGTZGTTTTGTZGTT- 3′ODN 8 5′-TCCAGGACTTCTCTCAGGTT-3′ ODN 9 5′-TCTCCCAGCGTGCGCCAT-3′ODN 10 murine 5′-TGCATCCCCCAGGCCACCAT-3 IntracellularAdhesion Molecule-1 ODN 11 human 5′-GCCCAAGCTGGCATCCGTCA-3′Intracellular Adhesion Molecule-1 ODN 12 human5′-GCCCAAGCTGGCATCCGTCA-3′ Intracellular Adhesion Molecule-1ODN 13 human erb-B-2 5′-GGT GCTCACTGC GGC-3′ ODN 14 human c-myc5′-AACC GTT GAG GGG CAT-3′ ODN 15 human c-myc5′-TAT GCT GTG CCG GGG TCT TCG GGC-3′ ODN 16 5′-GTGCCG GGGTCTTCGGGC-3′ODN 17 human Insulin 5′-GGACCCTCCTCCGGAGCC-3′ Growth Factor 1 - ReceptorODN 18 human Insulin 5′-TCC TCC GGA GCC AGA CTT-3′Growth Factor 1 - Receptor ODN 19 human Epidermal5′-AAC GTT GAG GGG CAT-3′ Growth Factor - ReceptorODN 20 Epidermal Growth 5′-CCGTGGTCA TGCTCC-3′ Factor - ReceptorODN 21 human Vascular 5′-CAG CCTGGCTCACCG CCTTGG-3′Endothelial Growth Factor ODN 22 murine 5′-CAG CCA TGG TTC CCC CCA AC-Phosphokinase C - alpha 3′ ODN 23 5′-GTT CTC GCT GGT GAG TTT CA-3′ODN 24 human Bcl-2 5′-TCT CCCAGCGTGCGCCAT-3′ ODN 25 human C-Raf-s5′-GTG CTC CAT TGA TGC-3′ ODN #26 human Vascular 5′- Endothelial GrowthGAGUUCUGAUGAGGCCGAAAGG- Factor Receptor-1 CCGAAAGUCUG-3′ ODN #275′-RRCGYY-3′ ODN # 28 5′-AACGTTGAGGGGCAT-3′ ODN #295′-CAACGTTATGGGGAGA-3′ ODN #30 human c-myc 5′-TAACGTTGAGGGGCAT-3′ “Z”represents a methylated cytosine residue. ODN 14 is a 15-meroligonucleotide and ODN 1 is the same oligonucleotide having a thymidineadded onto the 5′ end making ODN 1 into a 16-mer. No difference inbiological activity between ODN 14 and ODN 1 has been detected and bothexhibit similar immunostimulatory activity (Mui et al., 2001)

Additional specific nucleic acid sequences of oligonucleotides (ODNs)suitable for use in the compositions and methods of the invention aredescribed in Raney et al., Journal of Pharmacology and ExperimentalTherapeutics, 298:1185-1192 (2001). In certain embodiments, ODNs used inthe compositions and methods of the present invention have aphosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

Decoy Oligonucleotides

Because transcription factors recognize their relatively short bindingsequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to upregulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides may be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety.

Supermir

A supermir refers to a single stranded, double stranded or partiallydouble stranded oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or both or modifications thereof, which hasa nucleotide sequence that is substantially identical to an miRNA andthat is antisense with respect to its target. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages and which contain at leastone non-naturally-occurring portion which functions similarly. Suchmodified or substituted oligonucleotides are preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. In a preferred embodiment, thesupermir does not include a sense strand, and in another preferredembodiment, the supermir does not self-hybridize to a significantextent. An supermir featured in the invention can have secondarystructure, but it is substantially single-stranded under physiologicalconditions. An supermir that is substantially single-stranded issingle-stranded to the extent that less than about 50% (e.g., less thanabout 40%, 30%, 20%, 10%, or 5%) of the supermir is duplexed withitself. The supermir can include a hairpin segment, e.g., sequence,preferably at the 3′ end can self hybridize and form a duplex region,e.g., a duplex region of at least 1, 2, 3, or 4 and preferably less than8, 7, 6, or n nucleotides, e.g., 5 nucleotides. The duplexed region canbe connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or 6dTs, e.g., modified dTs. In another embodiment the supermir is duplexedwith a shorter oligo, e.g., of 5, 6, 7, 8, 9, or 10 nucleotides inlength, e.g., at one or both of the 3′ and 5′ end or at one end and inthe non-terminal or middle of the supermir.

miRNA mimics

miRNA mimics represent a class of molecules that can be used to imitatethe gene silencing ability of one or more miRNAs. Thus, the term“microRNA mimic” refers to synthetic non-coding RNAs (i.e. the miRNA isnot obtained by purification from a source of the endogenous miRNA) thatare capable of entering the RNAi pathway and regulating gene expression.miRNA mimics can be designed as mature molecules (e.g. single stranded)or mimic precursors (e.g., or pre-miRNAs). miRNA mimics can be comprisedof nucleic acid (modified or modified nucleic acids) includingoligonucleotides comprising, without limitation, RNA, modified RNA, DNA,modified DNA, locked nucleic acids, or 2′-O,4′-C-ethylene-bridgednucleic acids (ENA), or any combination of the above (including DNA-RNAhybrids). In addition, miRNA mimics can comprise conjugates that canaffect delivery, intracellular compartmentalization, stability,specificity, functionality, strand usage, and/or potency. In one design,miRNA mimics are double stranded molecules (e.g., with a duplex regionof between about 16 and about 31 nucleotides in length) and contain oneor more sequences that have identity with the mature strand of a givenmiRNA. Modifications can comprise 2′ modifications (including 2′-Omethyl modifications and 2′ F modifications) on one or both strands ofthe molecule and internucleotide modifications (e.g. phorphorthioatemodifications) that enhance nucleic acid stability and/or specificity.In addition, miRNA mimics can include overhangs. The overhangs canconsist of 1-6 nucleotides on either the 3′ or 5′ end of either strandand can be modified to enhance stability or functionality. In oneembodiment, a miRNA mimic comprises a duplex region of between 16 and 31nucleotides and one or more of the following chemical modificationpatterns: the sense strand contains 2′-O-methyl modifications ofnucleotides 1 and 2 (counting from the 5′ end of the senseoligonucleotide), and all of the Cs and Us; the antisense strandmodifications can comprise Z F modification of all of the Cs and Us,phosphorylation of the 5′ end of the oligonucleotide, and stabilizedinternucleotide linkages associated with a 2 nucleotide 3′ overhang.

Antimir or miRNA Inhibitor

The terms “antimir” “microRNA inhibitor”, “miR inhibitor”, or“inhibitor” are synonymous and refer to oligonucleotides or modifiedoligonucleotides that interfere with the ability of specific miRNAs. Ingeneral, the inhibitors are nucleic acid or modified nucleic acids innature including oligonucleotides comprising RNA; modified RNA, DNA,modified DNA, locked nucleic acids (LNAs), or any combination of theabove. Modifications include 2′ modifications (including 2′-0 alkylmodifications and 2′ F modifications) and internucleotide modifications(e.g. phosphorothioate modifications) that can affect delivery,stability, specificity, intracellular compartmentalization, or potency.In addition, miRNA inhibitors can comprise conjugates that can affectdelivery, intracellular compartmentalization, stability, and/or potency.Inhibitors can adopt a variety of configurations including singlestranded, double stranded (RNA/RNA or RNA/DNA duplexes), and hairpindesigns, in general, microRNA inhibitors comprise contain one or moresequences or portions of sequences that are complementary or partiallycomplementary with the mature strand (or strands) of the miRNA to betargeted, in addition, the miRNA inhibitor may also comprise additionalsequences located 5′ and 3′ to the sequence that is the reversecomplement of the mature miRNA. The additional sequences may be thereverse complements of the sequences that are adjacent to the maturemiRNA in the pri-miRNA from which the mature miRNA is derived, or theadditional sequences may be arbitrary sequences (having a mixture of A,G, C, or U). In some embodiments, one or both of the additionalsequences are arbitrary sequences capable of forming hairpins. Thus, insome embodiments, the sequence that is the reverse complement of themiRNA is flanked on the 5′ side and on the 3′ side by hairpinstructures. Micro-RNA inhibitors, when double stranded, may includemismatches between nucleotides on opposite strands. Furthermore,micro-RNA inhibitors may be linked to conjugate moieties in order tofacilitate uptake of the inhibitor into a cell. For example, a micro-RNAinhibitor may be linked to cholesteryl5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3 hydroxypentylcarbamate) whichallows passive uptake of a micro-RNA inhibitor into a cell. Micro-RNAinhibitors, including hairpin miRNA inhibitors, are described in detailin Vermeulen et al., “Double-Stranded Regions Are Essential DesignComponents Of Potent Inhibitors of RISC Function,” RNA 13: 723-730(2007) and in WO2007/095387 and WO 2008/036825 each of which isincorporated herein by reference in its entirety. A person of ordinaryskill in the art can select a sequence from the database for a desiredmiRNA and design an inhibitor useful for the methods disclosed herein.

U1 Adaptor

U1 adaptor inhibit polyA sites and are bifunctional oligonucleotideswith a target domain complementary to a site in the target gene'sterminal exon and a ‘U1 domain’ that binds to the U1 smaller nuclear RNAcomponent of the U1 snRNP (Goraczniak, et al., 2008, NatureBiotechnology, 27(3), 257-263, which is expressly incorporated byreference herein, in its entirety). U1 snRNP is a ribonucleoproteincomplex that functions primarily to direct early steps in spliceosomeformation by binding to the pre-mRNA exon-intron boundary (Brown andSimpson, 1998, Annu Rev Plant Physiol Plant MoI Biol 49:77-95).Nucleotides 2-11 of the 5′ end of U1 snRNA base pair bind with the 5′ssof the pre mRNA. In one embodiment, oligonucleotides of the inventionare U1 adaptors. In one embodiment, the U1 adaptor can be administeredin combination with at least one other iRNA agent.

Oligonucleotide Modifications

Unmodified oligonucleotides may be less than optimal in someapplications, e.g., unmodified oligonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications ofoligonucleotides can confer improved properties, and, e.g., can renderoligonucleotides more stable to nucleases.

As oligonucleotides are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin an oligonucleotide, e.g., a modification of a base, a sugar, aphosphate moiety, or the non-bridging oxygen of a phosphate moiety. Itis not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even ata single nucleoside within an oligonucleotide.

In some cases the modification will occur at all of the subjectpositions in the oligonucleotide but in many, and in fact in most casesit will not. By way of example, a modification may only occur at a 3′ or5′ terminal position, may only occur in the internal region, may onlyoccur in a terminal regions, e.g. at a position on a terminal nucleotideor in the last 2, 3, 4, 5, or 10 nucleotides of an oligonucleotide. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of a double-stranded oligonucleotide or may only occur in asingle strand region of a double-stranded oligonucleotide. E.g., aphosphorothioate modification at a non-bridging oxygen position may onlyoccur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. The 5′ end or ends can bephosphorylated.

A modification described herein may be the sole modification, or thesole type of modification included on multiple nucleotides, or amodification can be combined with one or more other modificationsdescribed herein. The modifications described herein can also becombined onto an oligonucleotide, e.g. different nucleotides of anoligonucleotide have different modifications described herein.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular nucleobases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-bridging oxygen atoms. However, thephosphate group can be modified by replacing one of the oxygens with adifferent substituent. One result of this modification to RNA phosphatebackbones can be increased resistance of the oligoribonucleotide tonucleolytic breakdown. Thus while not wishing to be bound by theory, itcan be desirable in some embodiments to introduce alterations whichresult in either an uncharged linker or a charged linker withunsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. In certain embodiments, one of the non-bridgingphosphate oxygen atoms in the phosphate backbone moiety can be replacedby any of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl), C(i.e. an alkyl group, an aryl group, etc. . . . ), H, NR₂ (R ishydrogen, alkyl, aryl), or OR (R is alkyl or aryl). The phosphorous atomin an unmodified phosphate group is achiral. However, replacement of oneof the non-bridging oxygens with one of the above atoms or groups ofatoms renders the phosphorous atom chiral; in other words a phosphorousatom in a phosphate group modified in this way is a stereogenic center.The stereogenic phosphorous atom can possess either the “R”configuration (herein Rp) or the “S” configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur.The phosphorus center in the phosphorodithioates is achiral whichprecludes the formation of oligoribonucleotides diastereomers. Thus,while not wishing to be bound by theory, modifications to bothnon-bridging oxygens, which eliminate the chiral center, e.g.phosphorodithioate formation, may be desirable in that they cannotproduce diastereomer mixtures. Thus, the non-bridging oxygens can beindependently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either linking oxygen or at both the linking oxygens. When thebridging oxygen is the 3′-oxygen of a nucleoside, replcament withcarbobn is preferred. When the bridging oxygen is the 5′-oxygen of anucleoside, replcament with nitrogen is preferred.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors. While not wishing to be bound by theory, it is believed thatsince the charged phosphodiester group is the reaction center innucleolytic degradation, its replacement with neutral structural mimicsshould impart enhanced nuclease stability. Again, while not wishing tobe bound by theory, it can be desirable, in some embodiment, tointroduce alterations in which the charged phosphate group is replacedby a neutral moiety.

Examples of moieties which can replace the phosphate group includemethyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include themethylenecarbonylamino and methylenemethylimino groups.

Modified phosphate linkages where at least one of the oxygens linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non phosphodiesterbackbone linkage.”

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. While not wishing to be bound bytheory, it is believed that the absence of a repetitively chargedbackbone diminishes binding to proteins that recognize polyanions (e.g.nucleases). Again, while not wishing to be bound by theory, it can bedesirable in some embodiment, to introduce alterations in which thebases are tethered by a neutral surrogate backbone. Examples include themophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA)nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Sugar Modifications

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a2′-alkoxide ion. The 2′-alkoxide can catalyze degradation byintramolecular nucleophilic attack on the linker phosphorus atom. Again,while not wishing to be bound by theory, it can be desirable to someembodiments to introduce alterations in which alkoxide formation at the2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH3,2′-O-allyl, 2′-C-allyl, and2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, an oligonucleotide can include nucleotidescontaining e.g., arabinose, as the sugar. The monomer can have an alphalinkage at the 1′ position on the sugar, e.g., alpha-nucleosides.Oligonucleotides can also include “abasic” sugars, which lack anucleobase at C-1′. These abasic sugars can also be further containingmodifications at one or more of the constituent sugar atoms.Oligonucleotides can also contain one or more sugars that are in the Lform, e.g. L-nucleosides.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a linker. The terminal atom of the linker canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the linkercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs).

When a linker/phosphate-functional molecular entity-linker/phosphatearray is interposed between two strands of a dsRNA, this array cansubstitute for a hairpin RNA loop in a hairpin-type RNA agent.

Terminal modifications useful for modulating activity includemodification of the 5′ end with phosphate or phosphate analogs. E.g., inpreferred embodiments antisense strands of dsRNAs, are 5′ phosphorylatedor include a phosphoryl analog at the 5′ prime terminus. 5′-phosphatemodifications include those which are compatible with RISC mediated genesilencing. Suitable modifications include: 5′-monophosphate((HO)₂(O)P—O-5′); 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap(7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate(phosphorothioate; (HO)₂(S)P—O-5); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Nucleobases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases”, “modified bases”, “non-natual bases” and“universal bases” described herein, can be employed. Examples includewithout limitation 2-aminoadenine, 6-methyl and other alkyl derivativesof adenine and guanine, 2-propyl and other alkyl derivatives of adenineand guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Cationic Groups

Modifications to oligonucleotides can also include attachment of one ormore cationic groups to the sugar, base, and/or the phosphorus atom of aphosphate or modified phosphate backbone moiety. A cationic group can beattached to any atom capable of substitution on a natural, unusual oruniversal base. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n) AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Placement within an Oligonucleotide

Some modifications may preferably be included on an oligonucleotide at aparticular location, e.g., at an internal position of a strand, or onthe 5′ or 3′ end of an oligonucleotide. A preferred location of amodification on an oligonucleotide, may confer preferred properties onthe agent. For example, preferred locations of particular modificationsmay confer optimum gene silencing properties, or increased resistance toendonuclease or exonuclease activity.

One or more nucleotides of an oligonucleotide may have a 2′-5′ linkage.One or more nucleotides of an oligonucleotide may have invertedlinkages, e.g. 3′-3′,5′-5′,2′-2′ or 2′-3′ linkages.

A double-stranded oligonucleotide may include at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine is a2′-modified nucleotide, or a terminal 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide, or aterminal 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide, or a terminal5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide, or a terminal 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide, or a terminal 5′-cytidine-uridine-3′ (5′-CU-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide, or aterminal 5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide. Double-stranded oligonucleotidesincluding these modifications are particularly stabilized againstendonuclease activity.

General References

The oligoribonucleotides and oligoribonucleotides used in accordancewith this invention may be synthesized with solid phase synthesis, seefor example “Oligonucleotide synthesis, a practical approach”, Ed. M. J.Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A PracticalApproach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1,Modern machine-aided methods of oligodeoxyribonucleotide synthesis,Chapter 2, Oligoribonucleotide synthesis, Chapter3,2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein. Modification described inWO 00/44895, WO01/75164, or WO02/44321 can be used herein. Thedisclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, et al. J. Org. Chem. 2001, 66,2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 11972, 1991. Carbamate replacements are described in Stirchak, E.P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Nucleobases References

N-2 substitued purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.

Linkers

The term “linker” means an organic moiety that connects two parts of acompound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynythereroaryl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R¹)₂,C(O), cleavable linking group, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclic; where R¹ is hydrogen, acyl, aliphatic or substitutedaliphatic.

In one embodiment, the linker is—[(P-Q-R)_(q)—X—(P′-Q′-R′)_(q′)]_(q″)-T-, wherein:

P, R, T, P′, R′ and T are each independently for each occurrence absent,CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, CH═N—O,

or heterocyclyl;

Q and Q′ are each independently for each occurrence absent, —(CH₂)_(n)—,—C(R¹)(R²)(CH₂)_(n)—, —(CH₂)_(n)C(R¹)(R²)—, —(CH₂CH₂O)_(m)CH₂CH₂—, or—(CH₂CH₂O)_(m)CH₂CH₂NH—;

X is absent or a cleavable linking group;

R^(a) is H or an amino acid side chain;

R¹ and R² are each independently for each occurrence H, CH₃, OH, SH orN(R^(N))₂;

R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q, q′ and q″ are each independently for each occurrence 0-20 and whereinthe repeating unit can be the same or different;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linkinggroup.

In certain embodiments, the linker is a branched linker. The branchpointof the branched linker may be at least trivalent, but may be atetravalent, pentavalent or hexavalent atom, or a group presenting suchmultiple valencies. In certain embodiments, the branchpoint is, —N,—N(Q)-C, —O—C, —S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, —N(Q)C(O)—C, or—N(Q)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum). Cleavable linkinggroups are susceptible to cleavage agents, e.g., pH, redox potential orthe presence of degradative molecules. Generally, cleavage agents aremore prevalent or found at higher levels or activities inside cells thanin serum or blood. Examples of such degradative agents include: redoxagents which are selected for particular substrates or which have nosubstrate specificity, including, e.g., oxidative or reductive enzymesor reductive agents such as mercaptans, present in cells, that candegrade a redox cleavable linking group by reduction; esterases;endosomes or agents that can create an acidic environment, e.g., thosethat result in a pH of five or lower; enzymes that can hydrolyze ordegrade an acid cleavable linking group by acting as a general acid,peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the chargedlipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the charged lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

Ligands

A wide variety of entities can be coupled to the oligonucleotides andlipids of the present invention. Preferred moieties are ligands, whichare coupled, preferably covalently, either directly or indirectly via anintervening tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Ligands providing enhancedaffinity for a selected target are also termed targeting ligands.Preferred ligands for conjugation to the lipids of the present inventionare targeting ligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In certain embodiments, the endosomolytic ligand assumesits active conformation at endosomal pH. The “active” conformation isthat conformation in which the endosomolytic ligand promotes lysis ofthe endosome and/or transport of the composition of the invention, orits components, from the endosome to the cytoplasm of the cell.Exemplary endosomolytic ligands include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al.,J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments,the endosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched. Exemplary primary sequences of peptide based endosomolyticligands are shown in Table 4.

TABLE 4 List of peptides with endosomolytic activity. NameSequence (N to C) Ref. GALA AALEALAEALEALAEALEALAEAAAAGGC 1 EALAAALAEALAEALAEALAEALAEALAAAAGGC 2 ALEALAEALEALAEA 3 INF-7GLFEAIEGFIENGWEGMEWDYG 4 Inf HA-2 GLFGAIAGFIENGWEGMIDGWYG 5 diINF-7GLF EAI EGFI ENGW EGMI DGWYGC 5 GLF EAI EGFI ENGW EGMI DGWYGC diINF3GLF EAI EGFI ENGW EGMI DGGC 6 GLF EAI EGFI ENGW EGMI DGGC GLFGLFGALAEALAEALAEHLAEALAEALEALAAGGSC 6 GALA-INF3GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC 6 INF-5 GLF EAI EGFI ENGW EGnI DG K 4GLF EAI EGFI ENGW EGnI DG n, norleucine References 1. Subbarao et al.,Biochemistry, 1987, 26: 2964-2972. 2. Vogel et al., J. Am. Chem. Soc.,1996, 118: 1581-1586 3. Turk, M. J., Reddy, J. A. et al. (2002).Characterization of a novel pH-sensitive peptide that enhances drugrelease from folate-targeted liposomes at endosomal pHs. Biochim.Biophys. Acta 1559, 56-68. 4. Plank, C. Oberhauser, B. Mechtler, K.Koch, C. Wagner, E. (1994). The influence of endosome-disruptivepeptides on gene transfer using synthetic virus-like gene transfersystems, J. Biol. Chem. 269 12918-12924. 5. Mastrobattista, E., Koning,G. A. et al. (2002). Functional characterization of anendosome-disruptive peptide and its application in cytosolic delivery ofimmunoliposome-entrapped proteins. J. Biol. Chem. 277, 27135-43. 6.Oberhauser, B., Plank, C. et al. (1995). Enhancing endosomal exit ofnucleic acids using pH-sensitive viral fusion peptides. Deliv.Strategies Antisense Oligonucleotide Ther. 247-66.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, charged lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 5 shows some examples of targetingligands and their associated receptors.

TABLE 5 Targeting Ligands and their associated receptors Liver CellsLigand Receptor 1) Parenchymal Galactose ASGP-R Cell (PC)(Asiologlycoprotein (Hepatocytes) receptor) Gal NAc ASPG-R(n-acetyl-galactosamine) Gal NAc Receptor Lactose Asialofetuin ASPG-r 2)Sinusoidal Hyaluronan Hyaluronan receptor Endothelial Cell ProcollagenProcollagen receptor (SEC) Negatively charged molecules Scavengerreceptors Mannose Mannose receptors N-acetyl Glucosamine Scavengerreceptors Immunoglobulins Fc Receptor LPS CD14 Receptor Insulin Receptormediated transcytosis Transferrin Receptor mediated transcytosisAlbumins Non-specific Sugar-Albumin conjugates Mannose-6-phosphateMannose-6-phosphate receptor 3) Kupffer Cell Mannose Mannose receptors(KC) Fucose Fucose receptors Albumins Non-specific Mannose-albuminconjugates

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein. e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long (see Table 6, for example).

TABLE 6 Exemplary Cell Permeation Peptides. Cell Permeation PeptideAmino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKKDerossi et al., J. Biol. Chem. 269: 10444, 1994 Tat fragmentGRKKRRQRRRPPQC Vives et al., J. Biol. Chem., (48-60) 272: 16010, 1997Signal Sequence- GALFLGWLGAAGSTMGAWSQPKKKR Chaloin et al., Biochem.based peptide KV Biophys. Res. Commun., 243: 601, 1998 PVECLLIILRRRIRKQAHAHSK Elmquist et al., Exp. Cell Res., 269: 237, 2001Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et al., FASEB J.,12: 67, 1998 Amphiphilic KLALKLALKALKAALKLA Oehlke et al., Mol. Ther.,model peptide 2: 339, 2000 Arg₉ RRRRRRRRR Mitchell et al., J. Pept. Res., 56: 318, 2000 Bacterial cell  KFFKFFKFFK wall permeating LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFL RNLVPRTES Cecropin P1SWLSKTAKKLENSAKKRISEGIAIAIQG GPR α-defensin ACYCRIPACIAGERRYGTCIYQGRLWAFCC b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTC YRGKAKCCK BactenecinRKCRIVV1RVCR PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGF PPRFPPRFPGKR-NH2Indolicidin ILPWKWPWWPWRR-NH2

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acid sequenceAALLPVLLAAP) containing a hydrophobic MTS can also be a targetingmoiety. The peptide moiety can be a “delivery” peptide, which can carrylarge polar molecules including peptides, oligonucleotides, and proteinacross cell membranes. For example, sequences from the HIV Tat protein(GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein(RQIKIWFQNRRMKWKK) have been found to be capable of functioning asdelivery peptides. A peptide or peptidomimetic can be encoded by arandom sequence of DNA, such as a peptide identified from aphage-display library, or one-bead-one-compound (OBOC) combinatoriallibrary (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide orpeptidomimetic tethered to an iRNA agent via an incorporated monomerunit is a cell targeting peptide such as an arginine-glycine-asparticacid (RGD)-peptide, or RGD mimic. A peptide moiety can range in lengthfrom about 5 amino acids to about 40 amino acids. The peptide moietiescan have a structural modification, such as to increase stability ordirect conformational properties. Any of the structural modificationsdescribed below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an αvβ3 integrin. Thus,one could use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the αvβ3 integrin ligand.Generally, such ligands can be used to control proliferating cells andangiogeneis. Preferred conjugates of this type lignads that targetsPECAM-1, VEGF, or other cancer gene, e.g., a cancer gene describedherein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an iRNA agent and/orthe carrier oligomer can be an amphipathic α-helical peptide. Exemplaryamphipathic α-helical peptides include, but are not limited to,cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide(BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfishintestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2,dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides,esculentinis-1, and caerins. A number of factors will preferably beconsidered to maintain the integrity of helix stability. For example, amaximum number of helix stabilization residues will be utilized (e.g.,leu, ala, or lys), and a minimum number helix destabilization residueswill be utilized (e.g., proline, or cyclic monomeric units. The cappingresidue will be considered (for example Gly is an exemplary N-cappingresidue and/or C-terminal amidation can be used to provide an extraH-bond to stabilize the helix. Formation of salt bridges betweenresidues with opposite charges, separated by i±3, or i±4 positions canprovide stability. For example, cationic residues such as lysine,arginine, homo-arginine, ornithine or histidine can form salt bridgeswith the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α,β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an apatamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPH, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Examplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbaone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligands amenable to the invention are described in copendingapplications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether. The ligand or tethered ligand may be present on amonomer when said monomer is incorporated into the growing strand. Insome embodiments, the ligand may be incorporated via coupling to a“precursor” monomer after said “precursor” monomer has been incorporatedinto the growing strand. For example, a monomer having, e.g., anamino-terminated tether (i.e., having no associated ligand), e.g.,TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisensestrand. In a subsequent operation, i.e., after incorporation of theprecursor monomer into the strand, a ligand having an electrophilicgroup, e.g., a pentafluorophenyl ester or aldehyde group, cansubsequently be attached to the precursor monomer by coupling theelectrophilic group of the ligand with the terminal nucleophilic groupof the precursor monomer's tether.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, lignad can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linking groups are well described in the literature such as,for example, in Manoharan in Antisense Research and Applications, Crookeand LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, whichis incorporated herein by reference in its entirety.

Representative United States patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,149,782; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662;5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434;6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631;6,559,279; each of which is herein incorporated by reference.

Characteristic of Nucleic Acid-Lipid Particles

In certain embodiments, the present invention relates to methods andcompositions for producing lipid-encapsulated nucleic acid particles inwhich nucleic acids are encapsulated within a lipid layer. Such nucleicacid-lipid particles, incorporating siRNA oligonucleotides, arecharacterized using a variety of biophysical parameters including: (1)drug to lipid ratio; (2) encapsulation efficiency; and (3) particlesize. High drug to lipid rations, high encapsulation efficiency, goodnuclease resistance and serum stability and controllable particle size,generally less than 200 nm in diameter are desirable. In addition, thenature of the nucleic acid polymer is of significance, since themodification of nucleic acids in an effort to impart nuclease resistanceadds to the cost of therapeutics while in many cases providing onlylimited resistance. Unless stated otherwise, these criteria arecalculated in this specification as follows:

Nucleic acid to lipid ratio is the amount of nucleic acid in a definedvolume of preparation divided by the amount of lipid in the same volume.This may be on a mole per mole basis or on a weight per weight basis, oron a weight per mole basis. For final, administration-readyformulations, the nucleic acid:lipid ratio is calculated after dialysis,chromatography and/or enzyme (e.g., nuclease) digestion has beenemployed to remove as much of the external nucleic acid as possible;

Encapsulation efficiency refers to the drug to lipid ratio of thestarting mixture divided by the drug to lipid ratio of the final,administration competent formulation. This is a measure of relativeefficiency. For a measure of absolute efficiency, the total amount ofnucleic acid added to the starting mixture that ends up in theadministration competent formulation, can also be calculated. The amountof lipid lost during the formulation process may also be calculated.Efficiency is a measure of the wastage and expense of the formulation;and

Size indicates the size (diameter) of the particles formed. Sizedistribution may be determined using quasi-elastic light scattering(QELS) on a Nicomp Model 370 sub-micron particle sizer. Particles under200 nm are preferred for distribution to neo-vascularized (leaky)tissues, such as neoplasms and sites of inflammation.

Pharmaceutical Compositions

The lipid particles of present invention, particularly when associatedwith a therapeutic agent, may beformulated as a pharmaceuticalcomposition, e.g., which further comprises a pharmaceutically acceptablediluent, excipient, or carrier, such as physiological saline orphosphate buffer, selected in accordance with the route ofadministration and standard pharmaceutical practice.

In particular embodiments, pharmaceutical compositions comprising thelipid-nucleic acid particles of the invention are prepared according tostandard techniques and further comprise a pharmaceutically acceptablecarrier. Generally, normal saline will be employed as thepharmaceutically acceptable carrier. Other suitable carriers include,e.g., water, buffered water, 0.9% saline, 0.3% glycine, and the like,including glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. In compositions comprising saline or othersalt containing carriers, the carrier is preferably added followinglipid particle formation. Thus, after the lipid-nucleic acidcompositions are formed, the compositions can be diluted intopharmaceutically acceptable carriers such as normal saline.

The resulting pharmaceutical preparations may be sterilized byconventional, well known sterilization techniques. The aqueous solutionscan then be packaged for use or filtered under aseptic conditions andlyophilized, the lyophilized preparation being combined with a sterileaqueous solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. Additionally, the lipidic suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as α-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The concentration of lipid particle or lipid-nucleic acid particle inthe pharmaceutical formulations can vary widely, i.e., from less thanabout 0.01%, usually at or at least about 0.05-5% to as much as 10 to30% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. For example, the concentration may be increasedto lower the fluid load associated with treatment. This may beparticularly desirable in patients having atherosclerosis-associatedcongestive heart failure or severe hypertension. Alternatively,complexes composed of irritating lipids may be diluted to lowconcentrations to lessen inflammation at the site of administration. Inone group of embodiments, the nucleic acid will have an attached labeland will be used for diagnosis (by indicating the presence ofcomplementary nucleic acid). In this instance, the amount of complexesadministered will depend upon the particular label used, the diseasestate being diagnosed and the judgement of the clinician but willgenerally be between about 0.01 and about 50 mg per kilogram of bodyweight, preferably between about 0.1 and about 5 mg/kg of body weight.

As noted above, the lipid-therapeutic agent (e.g., nucleic acid)particels of the invention may include polyethylene glycol(PEG)-modified phospholipids, PEG-ceramide, or gangliosideG_(M1)-modified lipids or other lipids effective to prevent or limitaggregation. Addition of such components does not merely prevent complexaggregation. Rather, it may also provide a means for increasingcirculation lifetime and increasing the delivery of the lipid-nucleicacid composition to the target tissues.

The present invention also provides lipid-therapeutic agent compositionsin kit form. The kit will typically be comprised of a container that iscompartmentalized for holding the various elements of the kit. The kitwill contain the particles or pharmaceutical compositions of the presentinvention, preferably in dehydrated or concentrated form, withinstructions for their rehydration or dilution and administration. Incertain embodiments, the particles comprise the active agent, while inother embodiments, they do not.

Methods of Manufacture

The methods and compositions of the invention make use of certaincharged lipids, the synthesis, preparation and characterization of whichis described below and in the accompanying Examples. In addition, thepresent invention provides methods of preparing lipid particles,including those associated with a therapeutic agent, e.g., a nucleicacid. In the methods described herein, a mixture of lipids is combinedwith a buffered aqueous solution of nucleic acid to produce anintermediate mixture containing nucleic acid encapsulated in lipidparticles wherein the encapsulated nucleic acids are present in anucleic acid/lipid ratio of about 3 wt % to about 25 wt %, preferably 5to 15 wt %. The intermediate mixture may optionally be sized to obtainlipid-encapsulated nucleic acid particles wherein the lipid portions areunilamellar vesicles, preferably having a diameter of 30 to 150 nm, morepreferably about 40 to 90 nm. The pH is then raised to neutralize atleast a portion of the surface charges on the lipid-nucleic acidparticles, thus providing an at least partially surface-neutralizedlipid-encapsulated nucleic acid composition.

As described above, several of these protonatable lipids are aminolipids that are charged at a pH below the pK_(a) of the amino group andsubstantially neutral at a pH above the pK_(a). These protonatablelipids are termed titratable cationic lipids and can be used in theformulations of the invention using a two-step process. First, lipidvesicles can be formed at the lower pH with titratable cationic lipidsand other vesicle components in the presence of nucleic acids. In thismanner, the vesicles will encapsulate and entrap the nucleic acids.Second, the surface charge of the newly formed vesicles can beneutralized by increasing the pH of the medium to a level above thepK_(a) of the titratable cationic lipids present, i.e., to physiologicalpH or higher. Particularly advantageous aspects of this process includeboth the facile removal of any surface adsorbed nucleic acid and aresultant nucleic acid delivery vehicle which has a neutral surface.Liposomes or lipid particles having a neutral surface are expected toavoid rapid clearance from circulation and to avoid certain toxicitieswhich are associated with cationic liposome preparations. Additionaldetails concerning these uses of such titratable cationic lipids in theformulation of nucleic acid-lipid particles are provided in U.S. Pat.No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporated herein byreference.

It is further noted that the vesicles formed in this manner provideformulations of uniform vesicle size with high content of nucleic acids.Additionally, the vesicles have a size range of from about 30 to about150 nm, more preferably about 30 to about 90 nm.

Without intending to be bound by any particular theory, it is believedthat the very high efficiency of nucleic acid encapsulation is a resultof electrostatic interaction at low pH. At acidic pH (e.g. pH 4.0) thevesicle surface is charged and binds a portion of the nucleic acidsthrough electrostatic interactions. When the external acidic buffer isexchanged for a more neutral buffer (e.g., pH 7.5) the surface of thelipid particle or liposome is neutralized, allowing any external nucleicacid to be removed. More detailed information on the formulation processis provided in various publications (e.g., U.S. Pat. No. 6,287,591 andU.S. Pat. No. 6,858,225).

In view of the above, the present invention provides methods ofpreparing lipid/nucleic acid formulations. In the methods describedherein, a mixture of lipids is combined with a buffered aqueous solutionof nucleic acid to produce an intermediate mixture containing nucleicacid encapsulated in lipid particles, e.g., wherein the encapsulatednucleic acids are present in a nucleic acid/lipid ratio of about 10 wt %to about 20 wt %. The intermediate mixture may optionally be sized toobtain lipid-encapsulated nucleic acid particles wherein the lipidportions are unilamellar vesicles, preferably having a diameter of 30 to150 mm, more preferably about 40 to 90 nm. The pH is then raised toneutralize at least a portion of the surface charges on thelipid-nucleic acid particles, thus providing an at least partiallysurface-neutralized lipid-encapsulated nucleic acid composition.

In certain embodiments, the mixture of lipids includes at least twolipid components: a first lipid component of the present invention thatis selected from among lipids which have a pKa such that the lipid iscationic at pH below the pKa and neutral at pH above the pKa, and asecond lipid component that is selected from among lipids that preventparticle aggregation during lipid-nucleic acid particle formation. Inparticular embodiments, the amino lipid is a novel charged lipid of thepresent invention.

In preparing the nucleic acid-lipid particles of the invention, themixture of lipids is typically a solution of lipids in an organicsolvent. This mixture of lipids can then be dried to form a thin film orlyophilized to form a powder before being hydrated with an aqueousbuffer to form liposomes. Alternatively, in a preferred method, thelipid mixture can be solubilized in a water miscible alcohol, such asethanol, and this ethanolic solution added to an aqueous bufferresulting in spontaneous liposome formation. In most embodiments, thealcohol is used in the form in which it is commercially available. Forexample, ethanol can be used as absolute ethanol (100%), or as 95%ethanol, the remainder being water. This method is described in moredetail in U.S. Pat. No. 5,976,567).

In one exemplary embodiment, the mixture of lipids is a mixture ofcharged lipids, neutral lipids (other than a charged lipid), a sterol(e.g., cholesterol) and a PEG-modified lipid (e.g., a PEG-DMG orPEG-DMA) in an alcohol solvent. In preferred embodiments, the lipidmixture consists essentially of a charged lipid, a neutral lipid,cholesterol and a PEG-modified lipid in alcohol, more preferablyethanol. In further preferred embodiments, the first solution consistsof the above lipid mixture in molar ratios of about 20-70% chargedlipid: 5-45% neutral lipid:20-55% cholesterol:0.5-15% PEG-modifiedlipid. In still further preferred embodiments, the first solutionconsists essentially of a lipid chosen from Table 1 or Table 2, DSPC,Chol and PEG-DMG or PEG-DMA, more preferably in a molar ratio of about20-60% charged lipid: 5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA.In particular embodiments, the molar lipid ratio is approximately40/10/40/10 (mol % charged lipid/DSPC/Chol/PEG-DMG or PEG-DMA),35/15/40/10 (mol % charged lipid/DSPC/Chol/PEG-DMG or PEG-DMA) or52/13/30/5 (mol % charged lipid/DSPC/Chol/PEG-DMG or PEG-DMA). Inanother group of preferred embodiments, the neutral lipid in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

In accordance with the invention, the lipid mixture is combined with abuffered aqueous solution that may contain the nucleic acids. Thebuffered aqueous solution of is typically a solution in which the bufferhas a pH of less than the pK_(a) of the protonatable lipid in the lipidmixture. Examples of suitable buffers include citrate, phosphate,acetate, and MES. A particularly preferred buffer is citrate buffer.Preferred buffers will be in the range of 1-1000 mM of the anion,depending on the chemistry of the nucleic acid being encapsulated, andoptimization of buffer concentration may be significant to achievinghigh loading levels (see, e.g., U.S. Pat. No. 6,287,591 and U.S. Pat.No. 6,858,225). Alternatively, pure water acidified to pH 5-6 withchloride, sulfate or the like may be useful. In this case, it may besuitable to add 5% glucose, or another non-ionic solute which willbalance the osmotic potential across the particle membrane when theparticles are dialyzed to remove ethanol, increase the pH, or mixed witha pharmaceutically acceptable carrier such as normal saline. The amountof nucleic acid in buffer can vary, but will typically be from about0.01 mg/mL to about 200 mg/mL, more preferably from about 0.5 mg/mL toabout 50 mg/mL.

The mixture of lipids and the buffered aqueous solution of therapeuticnucleic acids is combined to provide an intermediate mixture. Theintermediate mixture is typically a mixture of lipid particles havingencapsulated nucleic acids. Additionally, the intermediate mixture mayalso contain some portion of nucleic acids which are attached to thesurface of the lipid particles (liposomes or lipid vesicles) due to theionic attraction of the negatively-charged nucleic acids andpositively-charged lipids on the lipid particle surface (the aminolipids or other lipid making up the protonatable first lipid componentare positively charged in a buffer having a pH of less than the pK_(a)of the protonatable group on the lipid). In one group of preferredembodiments, the mixture of lipids is an alcohol solution of lipids andthe volumes of each of the solutions is adjusted so that uponcombination, the resulting alcohol content is from about 20% by volumeto about 45% by volume. The method of combining the mixtures can includeany of a variety of processes, often depending upon the scale offormulation produced. For example, when the total volume is about 10-20mL or less, the solutions can be combined in a test tube and stirredtogether using a vortex mixer. Large-scale processes can be carried outin suitable production scale glassware.

Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleicacid) complexes which are produced by combining the lipid mixture andthe buffered aqueous solution of therapeutic agents (nucleic acids) canbe sized to achieve a desired size range and relatively narrowdistribution of lipid particle sizes. Preferably, the compositionsprovided herein will be sized to a mean diameter of from about 70 toabout 200 nm, more preferably about 90 to about 130 nm. Severaltechniques are available for sizing liposomes to a desired size. Onesizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles (SUVs) less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both methods, the particlesize distribution can be monitored by conventional laser-beam particlesize determination. For certain methods herein, extrusion is used toobtain a uniform vesicle size.

Extrusion of liposome compositions through a small-pore polycarbonatemembrane or an asymmetric ceramic membrane results in a relativelywell-defined size distribution. Typically, the suspension is cycledthrough the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing.

In particular embodiments, methods of the present invention furthercomprise a step of neutralizing at least some of the surface charges onthe lipid portions of the lipid-nucleic acid compositions. By at leastpartially neutralizing the surface charges, unencapsulated nucleic acidis freed from the lipid particle surface and can be removed from thecomposition using conventional techniques. Preferably, unencapsulatedand surface adsorbed nucleic acids are removed from the resultingcompositions through exchange of buffer solutions. For example,replacement of a citrate buffer (pH about 4.0, used for forming thecompositions) with a HEPES-buffered saline (FIBS pH about 7.5) solution,results in the neutralization of liposome surface and nucleic acidrelease from the surface. The released nucleic acid can then be removedvia chromatography using standard methods, and then switched into abuffer with a pH above the pKa of the lipid used.

Optionally the lipid vesicles (i.e., lipid particles) can be formed byhydration in an aqueous buffer and sized using any of the methodsdescribed above prior to addition of the nucleic acid. As describedabove, the aqueous buffer should be of a pH below the pKa of the aminolipid. A solution of the nucleic acids can then be added to these sized,preformed vesicles. To allow encapsulation of nucleic acids into such“pre-formed” vesicles the mixture should contain an alcohol, such asethanol. In the case of ethanol, it should be present at a concentrationof about 20% (w/w) to about 45% (w/w). In addition, it may be necessaryto warm the mixture of pre-formed vesicles and nucleic acid in theaqueous buffer-ethanol mixture to a temperature of about 25° C. to about50° C. depending on the composition of the lipid vesicles and the natureof the nucleic acid. It will be apparent to one of ordinary skill in theart that optimization of the encapsulation process to achieve a desiredlevel of nucleic acid in the lipid vesicles will require manipulation ofvariable such as ethanol concentration and temperature. Examples ofsuitable conditions for nucleic acid encapsulation are provided in theExamples, Once the nucleic acids are encapsulated within the prefromedvesicles, the external pH can be increased to at least partiallyneutralize the surface charge. Unencapsulated and surface adsorbednucleic acids can then be removed as described above.

Method of Use

The lipid particles of the present invention may be used to deliver atherapeutic agent to a cell, in vitro or in vivo. In particularembodiments, the therapeutic agent is a nucleic acid, which is deliveredto a cell using a nucleic acid-lipid particles of the present invention.While the following description o various methods of using the lipidparticles and related pharmaceutical compositions of the presentinvention are exemplified by description related to nucleic acid-lipidparticles, it is understood that these methods and compositions may bereadily adapted for the delivery of any therapeutic agent for thetreatment of any disease or disorder that would benefit from suchtreatment.

In certain embodiments, the present invention provides methods forintroducing a nucleic acid into a cell. Preferred nucleic acids forintroduction into cells are siRNA, immune-stimulating oligonucleotides,plasmids, antisense and ribozymes. These methods may be carried out bycontacting the particles or compositions of the present invention withthe cells for a period of time sufficient for intracellular delivery tooccur.

The compositions of the present invention can be adsorbed to almost anycell type. Once adsorbed, the nucleic acid-lipid particles can either beendocytosed by a portion of the cells, exchange lipids with cellmembranes, or fuse with the cells. Transfer or incorporation of thenucleic acid portion of the complex can take place via any one of thesepathways. Without intending to be limited with respect to the scope ofthe invention, it is believed that in the case of particles taken upinto the cell by endocytosis the particles then interact with theendosomal membrane, resulting in destabilization of the endosomalmembrane, possibly by the formation of non-bilayer phases, resulting inintroduction of the encapsulated nucleic acid into the cell cytoplasm.Similarly in the case of direct fusion of the particles with the cellplasma membrane, when fusion takes place, the liposome membrane isintegrated into the cell membrane and the contents of the liposomecombine with the intracellular fluid. Contact between the cells and thelipid-nucleic acid compositions, when carried out in vitro, will takeplace in a biologically compatible medium. The concentration ofcompositions can vary widely depending on the particular application,but is generally between about 1 μmol and about 10 mmol. In certainembodiments, treatment of the cells with the lipid-nucleic acidcompositions will generally be carried out at physiological temperatures(about 37° C.) for periods of time from about 1 to 24 hours, preferablyfrom about 2 to 8 hours. For in vitro applications, the delivery ofnucleic acids can be to any cell grown in culture, whether of plant oranimal origin, vertebrate or invertebrate, and of any tissue or type. Inpreferred embodiments, the cells will be animal cells, more preferablymammalian cells, and most preferably human cells.

In one group of embodiments, a lipid-nucleic acid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/mL, more preferably about 2×10⁴ cells/mL.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 20 μg/mL, more preferably about 1 μg/mL.

In another embodiment, the lipid particles of the invention can be maybe used to deliver a nucleic acid to a cell or cell line (for example, atumor cell line). Non-limiting examples of such cell lines include: HELA(ATCC Cat N: CCL-2), KB (ATCC Cat N: CCL-17), HEP3B (ATCC Cat N:HB-8064), SKOV-3 (ATCC Cat N: HTB-77), HCT-116 (ATCC Cat N: CCL-247),HT-29 (ATCC Cat N: HTB-38), PC-3 (ATCC Cat N: CRL-1435), A549 (ATCC CatN: CCL-185), MDA-MB-231 (ATCC Cat N: HTB-26).

Typical applications include using well known procedures to provideintracellular delivery of siRNA to knock down or silence specificcellular targets. Alternatively applications include delivery of DNA ormRNA sequences that code for therapeutically useful polypeptides. Inthis manner, therapy is provided for genetic diseases by supplyingdeficient or absent gene products (i.e., for Duchenne's dystrophy, seeKunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989), and for cysticfibrosis, see Goodfellow, Nature 341:102-103 (1989)). Other uses for thecompositions of the present invention include introduction of antisenseoligonucleotides in cells (see, Bennett, et al., Mol. Pharm.41:1023-1033 (1992)).

Alternatively, the compositions of the present invention can also beused for deliver of nucleic acids to cells in vivo, using methods whichare known to those of skill in the art. With respect to delivery of DNAor mRNA sequences, au, et al., Science 261:209-211 (1993), incorporatedherein by reference, describes the intravenous delivery ofcytomegalovirus (CMV)-chloramphenicol acetyltransferase (CAT) expressionplasmid using DOTMA-DOPE complexes. Hyde, et al., Nature 362:250-256(1993), incorporated herein by reference, describes the delivery of thecystic fibrosis transmembrane conductance regulator (CFTR) gene toepithelia of the airway and to alveoli in the lung of mice, usingliposomes. Brigham, et al., Am. J. Med. Sci. 298:278-281 (1989),incorporated herein by reference, describes the in vivo transfection oflungs of mice with a functioning prokaryotic gene encoding theintracellular enzyme, chloramphenicol acetyltransferase (CAT). Thus, thecompositions of the invention can be used in the treatment of infectiousdiseases.

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally, i.e., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly. Inparticular embodiments, the pharmaceutical compositions are administeredintravenously or intraperitoneally by a bolus injection. For oneexample, see Stadler, et al., U.S. Pat. No. 5,286,634, which isincorporated herein by reference. Intracellular nucleic acid deliveryhas also been discussed in Straubringer, et al., METHODS IN ENZYMOLOGY,Academic Press, New York 101:512-527 (1983); Mannino, et al.,Biotechniques 6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. DrugCarrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278(1993). Still other methods of administering lipid-based therapeuticsare described in, for example, Rahman et al., U.S. Pat. No. 3,993,754;Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No.4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat.No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

In other methods, the pharmaceutical preparations may be contacted withthe target tissue by direct application of the preparation to thetissue. The application may be made by topical, “open” or “closed”procedures. By “topical,” it is meant the direct application of thepharmaceutical preparation to a tissue exposed to the environment, suchas the skin, oropharynx, external auditory canal, and the like. “Open”procedures are those procedures which include incising the skin of apatient and directly visualizing the underlying tissue to which thepharmaceutical preparations are applied. This is generally accomplishedby a surgical procedure, such as a thoracotomy to access the lungs,abdominal laparotomy to access abdominal viscera, or other directsurgical approach to the target tissue. “Closed” procedures are invasiveprocedures in which the internal target tissues are not directlyvisualized, but accessed via inserting instruments through small woundsin the skin. For example, the preparations may be administered to theperitoneum by needle lavage. Likewise, the pharmaceutical preparationsmay be administered to the meninges or spinal cord by infusion during alumbar puncture followed by appropriate positioning of the patient ascommonly practiced for spinal anesthesia or metrizamide imaging of thespinal cord. Alternatively, the preparations may be administered throughendoscopic devices.

The lipid-nucleic acid compositions can also be administered in anaerosol inhaled into the lungs (see, Brigham, et al., Am. J. Sci.298(4):278-281 (1989)) or by direct injection at the site of disease(Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, NewYork. pp. 70-71 (1994)).

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as humans,non-human primates, dogs, cats, cattle, horses, sheep, and the like.

Dosages for the lipid-therapeutic agent particles of the presentinvention will depend on the ratio of therapeutic agent to lipid and theadministrating physician's opinion based on age, weight, and conditionof the patient.

In one embodiment, the present invention provides a method of modulatingthe expression of a target polynucleotide or polypeptide. These methodsgenerally comprise contacting a cell with a lipid particle of thepresent invention that is associated with a nucleic acid capable ofmodulating the expression of a target polynucleotide or polypeptide. Asused herein, the term “modulating” refers to altering the expression ofa target polynucleotide or polypeptide. In different embodiments,modulating can mean increasing or enhancing, or it can mean decreasingor reducing. Methods of measuring the level of expression of a targetpolynucleotide or polypeptide are known and available in the arts andinclude, e.g., methods employing reverse transcription-polymerase chainreaction (RT-PCR) and immunohistochemical techniques. In particularembodiments, the level of expression of a target polynucleotide orpolypeptide is increased or reduced by at least 10%, 20%, 30%, 40%, 50%,or greater than 50% as compared to an appropriate control value.

For example, if increased expression of a polypeptide desired, thenucleic acid may be an expression vector that includes a polynucleotidethat encodes the desired polypeptide. On the other hand, if reducedexpression of a polynucleotide or polypeptide is desired, then thenucleic acid may be, e.g., an antisense oligonucleotide, siRNA, ormicroRNA that comprises a polynucleotide sequence that specificallyhybridizes to a polnucleotide that encodes the target polypeptide,thereby disrupting expression of the target polynucleotide orpolypeptide. Alternatively, the nucleic acid may be a plasmid thatexpresses such an antisense oligonucletoide, siRNA, or microRNA.

In one particular embodiment, the present invention provides a method ofmodulating the expression of a polypeptide by a cell, comprisingproviding to a cell a lipid particle that consists of or consistsessentially of a lipid chosen from Table 1 or Table 2, DSPC, Chol andPEG-DMG or PEG-DMA, e.g., in a molar ratio of about 20-60% chargedlipid: 5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA, wherein thelipid particle is assocated with a nucleic acid capable of modulatingthe expression of the polypeptide. In particular embodiments, the molarlipid ratio is approximately 40/10/40/10 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

In particular embodiments, the therapeutic agent is selected from ansiRNA, a microRNA, an antisense oligonucleotide, and a plasmid capableof expressing an siRNA, a microRNA, or an antisense oligonucleotide, andwherein the siRNA, microRNA, or antisense RNA comprises a polynucleotidethat specifically binds to a polynucleotide that encodes thepolypeptide, or a complement thereof, such that the expression of thepolypeptide is reduced.

In other embodiments, the nucleic acid is a plasmid that encodes thepolypeptide or a functional variant or fragment thereof, such thatexpression of the polypeptide or the functional variant or fragmentthereof is increased.

In related embodiments, the present invention provides a method oftreating a disease or disorder characterized by overexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is selected from an siRNA, a microRNA, an antisenseoligonucleotide, and a plasmid capable of expressing an siRNA, amicroRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of a lipid chosen fromTable 1 or Table 2, DSPC, Chol and PEG-DMG or PEG-DMA, e.g., in a molarratio of about 20-60% charged lipid: 5-25% DSPC:25-55% Chol:0.5-15%PEG-DMG or PEG-DMA, wherein the lipid particle is assocated with thetherapeutic nucleic acid. In particular embodiments, the molar lipidratio is approximately 40/10/40/10 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

In another related embodiment, the present invention includes a methodof treating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of a lipid chosen fromTable 1 or Table 2, DSPC, Chol and PEG-DMG or PEG-DMA, e.g., in a molarratio of about 20-60% charged lipid: 5-25% DSPC:25-55% Chol:0.5-15%PEG-DMG or PEG-DMA, wherein the lipid particle is assocated with thetherapeutic nucleic acid. In particular embodiments, the molar lipidratio is approximately 40/10/40/10 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

The present invention further provides a method of inducing an immuneresponse in a subject, comprising providing to the subject thepharmaceutical composition of the present invention, wherein thetherapeutic agent is an immunostimulatory oligonucleotide. In certainembodiments, the immune response is a humoral or mucosal immuneresponse. In one embodiment, the pharmaceutical composition comprises alipid particle that consists of or consists essentially of a lipidchosen from Table 1 or Table 2, DSPC, Chol and PEG-DMG or PEG-DMA, e.g.,in a molar ratio of about 20-60% charged lipid: 5-25% DSPC:25-55%Chol:0.5-15% PEG-DMG or PEG-DMA, wherein the lipid particle is assocatedwith the therapeutic nucleic acid. In particular embodiments, the molarlipid ratio is approximately 40/10/40/10 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % chargedlipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

In further embodiments, the pharmaceutical composition is provided tothe subject in combination with a vaccine or antigen. Thus, the presentinvention itself provides vaccines comprising a lipid particle of thepresent invention, which comprises an immunostimulatory oligonucleotide,and is also associated with an antigen to which an immune response isdesired. In particular embodiments, the antigen is a tumor antigen or isassociated with an infective agent, such as, e.g., a virus, bacteria, orparasiste.

A variety of tumor antigens, infections agent antigens, and antigensassociated with other disease are well known in the art and examples ofthese are described in references cited herein. Examples of antigenssuitable for use in the present invention include, but are not limitedto, polypeptide antigens and DNA antigens. Specific examples of antigensare Hepatitis A, Hepatitis B, small pox, polio, anthrax, influenza,typhus, tetanus, measles, rotavirus, diphtheria, pertussis,tuberculosis, and rubella antigens. In a preferred embodiment, theantigen is a Hepatitis B recombinant antigen. In other aspects, theantigen is a Hepatitis A recombinant antigen. In another aspect, theantigen is a tumor antigen. Examples of such tumor-associated antigensare MUC-1, EB V antigen and antigens associated with Burkitt's lymphoma.In a further aspect, the antigen is a tyrosinase-related protein tumorantigen recombinant antigen. Those of skill in the art will know ofother antigens suitable for use in the present invention.

Tumor-associated antigens suitable for use in the subject inventioninclude both mutated and non-mutated molecules that may be indicative ofsingle tumor type, shared among several types of tumors, and/orexclusively expressed or overexpressed in tumor cells in comparison withnormal cells. In addition to proteins and glycoproteins, tumor-specificpatterns of expression of carbohydrates, gangliosides, glycolipids andmucins have also been documented. Exemplary tumor-associated antigensfor use in the subject cancer vaccines include protein products ofoncogenes, tumor suppressor genes and other genes with mutations orrearrangements unique to tumor cells, reactivated embryonic geneproducts, oncofetal antigens, tissue-specific (but not tumor-specific)differentiation antigens, growth factor receptors, cell surfacecarbohydrate residues, foreign viral proteins and a number of other selfproteins.

Specific embodiments of tumor-associated antigens include, e.g., mutatedantigens such as the protein products of the Ras p21 protooncogenes,tumor suppressor p53 and BCR-abl oncogenes, as well as CDK4, MUM1,Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4,galectin 9, carbonic anhydrase, Aldolase A, PRAME, Her2/neu, ErbB-2 andKSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionicgonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA)and melanocyte differentiation antigens such as Mart 1/Melan A, gp100,gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such asPSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene productssuch as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE, RAGE, and othercancer testis antigens such as NY-ESO1, SSX2 and SCP1; mucins such asMuc-1 and Muc-2; gangliosides such as GM2, GD2 and GD3, neutralglycolipids and glycoproteins such as Lewis (y) and globo-H; andglycoproteins such as Tn, Thompson-Freidenreich antigen (TF) and sTn.Also included as tumor-associated antigens herein are whole cell andtumor cell lysates as well as immunogenic portions thereof, as well asimmunoglobulin idiotypes expressed on monoclonal proliferations of Blymphocytes for use against B cell lymphomas.

Pathogens include, but are not limited to, infectious agents, e.g.,viruses, that infect mammals, and more particularly humans. Examples ofinfectious virus include, but are not limited to: Retroviridae (e.g.,human immunodeficiency viruses, such as HIV-1 (also referred to asHTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g.,dengue viruses, encephalitis viruses, yellow fever viruses);Coronoviridae (e.g., coronaviruses); Rhabdoviradae (e.g., vesicularstomatitis viruses, rabies viruses); Coronaviridae (e.g.,coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae(e.g., parainfluenza viruses, mumps virus, measles virus, respiratorysyncytial virus); Orthomyxoviridae (e.g., influenza viruses);Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae(e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2,varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae(e.g., African swine fever virus); and unclassified viruses (e.g., theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses).

Also, gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfuenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Additional examples of pathogens include, but are not limited to,infectious fungi that infect mammals, and more particularly humans.Examples of infectious fingi include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.Examples of infectious parasites include Plasmodium such as Plasmodiumfalciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax.Other infectious organisms (i.e., protists) include Toxoplasma gondii.

In one embodiment, the formulations of the invention can be used tosilence or modulate a target gene such as but not limited to FVII, Eg5,PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene,CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin Dgene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivingene, Her2/Neu gene, SORT1 gene, XBP1 gene, topoisomerase I gene,topoisomerase II alpha gene, p73 gene, p21(WAF1/CIP1) gene, p27(KIP1)gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68gene, tumor suppressor genes, p53 tumor suppressor gene, p53 familymember DN-p63, pRb tumor suppressor gene, APC1 tumor suppressor gene,BRCA1 tumor suppressor gene, PTEN tumor suppressor gene, mLL fusiongene, BCR/ABL fusion gene, TEUAML1 fusion gene, EWS/FLI1 fusion gene,TLS/FUS1 fusion gene, PAX3/FKHR fusion gene, AML1/ETO fusion gene, alphav-integrin gene, Flt-1 receptor gene, tubulin gene, Human PapillomaVirus gene, a gene required for Human Papilloma Virus replication, HumanImmunodeficiency Virus gene, a gene required for Human ImmunodeficiencyVirus replication, Hepatitis A Virus gene, a gene required for HepatitisA Virus replication, Hepatitis B Virus gene, a gene required forHepatitis B Virus replication, Hepatitis C Virus gene, a gene requiredfor Hepatitis C Virus replication, Hepatitis D Virus gene, a generequired for Hepatitis D Virus replication, Hepatitis E Virus gene, agene required for Hepatitis E Virus replication, Hepatitis F Virus gene,a gene required for Hepatitis F Virus replication, Hepatitis G Virusgene, a gene required for Hepatitis G Virus replication, Hepatitis HVirus gene, a gene required for Hepatitis H Virus replication,Respiratory Syncytial Virus gene, a gene that is required forRespiratory Syncytial Virus replication, Herpes Simplex Virus gene, agene that is required for Herpes Simplex Virus replication, herpesCytomegalovirus gene, a gene that is required for herpes Cytomegalovirusreplication, herpes Epstein Barr Virus gene, a gene that is required forherpes Epstein Barr Virus replication, Kaposi's Sarcoma-associatedHerpes Virus gene, a gene that is required for Kaposi'sSarcoma-associated Herpes Virus replication, JC Virus gene, human genethat is required for JC Virus replication, myxovirus gene, a gene thatis required for myxovirus gene replication, rhinovirus gene, a gene thatis required for rhinovirus replication, coronavirus gene, a gene that isrequired for coronavirus replication, West Nile Virus gene, a gene thatis required for West Nile Virus replication, St. Louis Encephalitisgene, a gene that is required for St. Louis Encephalitis replication,Tick-borne encephalitis virus gene, a gene that is required forTick-borne encephalitis virus replication, Murray Valley encephalitisvirus gene, a gene that is required for Murray Valley encephalitis virusreplication, dengue virus gene, a gene that is required for dengue virusgene replication, Simian Virus 40 gene, a gene that is required forSimian Virus 40 replication, Human T Cell Lymphotropic Virus gene, agene that is required for Human T Cell Lymphotropic Virus replication,Moloney-Murine Leukemia Virus gene, a gene that is required forMoloney-Murine Leukemia Virus replication, encephalomyocarditis virusgene, a gene that is required for encephalomyocarditis virusreplication, measles virus gene, a gene that is required for measlesvirus replication, Vericella zoster virus gene, a gene that is requiredfor Vericella zoster virus replication, adenovirus gene, a gene that isrequired for adenovirus replication, yellow fever virus gene, a genethat is required for yellow fever virus replication, poliovirus gene, agene that is required for poliovirus replication, poxvirus gene, a genethat is required for poxvirus replication, plasmodium gene, a gene thatis required for plasmodium gene replication, Mycobacterium ulceransgene, a gene that is required for Mycobacterium ulcerans replication,Mycobacterium tuberculosis gene, a gene that is required forMycobacterium tuberculosis replication, Mycobacterium leprae gene, agene that is required for Mycobacterium leprae replication,Staphylococcus aureus gene, a gene that is required for Staphylococcusaureus replication, Streptococcus pneumoniae gene, a gene that isrequired for Streptococcus pneumoniae replication, Streptococcuspyogenes gene, a gene that is required for Streptococcus pyogenesreplication, Chlamydia pneumoniae gene, a gene that is required forChlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-11 gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, 1-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCAT gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene.

DEFINITIONS

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

The term “acyl” refers to hydrogen, alkyl, partially saturated or fullysaturated cycloalkyl, partially saturated or fully saturatedheterocycle, aryl, and heteroaryl substituted carbonyl groups. Forexample, acyl includes groups such as (C1-C20)alkanoyl (e.g., formyl,acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.),(C3-C20)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl,tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl(e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl,furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,benzo[b]thiophenyl-2-carbonyl, etc.).

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond. The term“alkylheterocyle” refers to a heteroaryl wherein at least one of thering atoms is substituted with alkyl, alkenyl or alkynyl

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio,alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl,arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl,haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino,alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl,carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl,aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonicacid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understoodthat the substituent may be further substituted. Exemplary substituentsinclude amino, alkylamino, dialkylamino, and cyclic amino compounds.

“Halogen” means fluoro, chloro, bromo and iodo.

The terms “alkylamine” and “dialkylamine” refer to —NH(alkyl) and —N(alkyl)₂ radicals respectively.

The term “alkylphosphate” refers to —O—P(Q′)(Q″)—O—R, wherein Q′ and Q″are each independently O, S, N(R)₂, optionally substituted alkyl oralkoxy; and R is optionally substituted alkyl, ω-aminoalkyl orω-(substituted)aminoalkyl.

The term “alkylphosphorothioate” refers to an alkylphosphate wherein atleast one of Q′ or Q″ is S.

The term “alkylphosphonate” refers to an alkylphosphate wherein at leastone of Q′ or Q″ is alkyl.

The terem “hydroxyalkyl” means —O-alkyl radical.

The term “alkylheterocycle” refers to an alkyl where at least onemethylene has been replaced by a heterocycle.

The term “ω-aminoalkyl” refers to -alkyl-NH₂ radical. And the term“ω-(substituted)aminoalkyl refers to an w-aminoalkyl wherein at leastone of the H on N has been replaced with alkyl.

The term “ω-phosphoalkyl” refers to -alkyl-O—P(Q′)(Q″)—O—R, wherein Q′and Q″ are each independently O or S and R optionally substituted alkyl.

The term “ω-thiophosphoalkyl refers to ω-phosphoalkyl wherein at leastone of Q′ or Q″ is S.

In some embodiments, the methods of the invention may require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANICSYNTHESIS, Green, T. W. et. al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

The compounds of the present invention may be prepared by known organicsynthesis techniques, including the methods described in more detail inthe Examples.

EXAMPLES Materials/Methods Synthesis of(3aR,5s,6aS)—N,N,N-trimethyl-2-[((9Z,12Z)-octadeca-9,12-dienyl)-2-octadecyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-aminium chloride L8

The method for synthesizing(3aR,5s,6aS)—N,N,N-trimethyl-2-((9Z,12Z)-octadeca-9,12-dienyl)-2-octadecyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-aminium chloride (L8) isbriefly described below.

Synthesis of linoleyl mesylate or (9Z,12Z)-octadeca-9,12-dienylmethanesulfonate (2)

Referring to Scheme 10, triethylamine (13.13 g, 130 mmol) was added to asolution of the linoleyl alcohol 1 (26.6 g, 100 mmol) in dichloromethane(100 mL) and the solution was cooled in an ice-bath. To this coldsolution, a solution of methanesulfonyl chloride (12.6 g, 110 mmol) indichloromethane (60 mL) was added dropwise; after the completion of theaddition, the reaction mixture was allowed to warm to ambienttemperature and stirred overnight. Completion of the reaction wasconfirmed by TLC. The reaction mixture was diluted with dichloromethane(200 mL), washed with water (200 mL), satd. NaHCO₃ (200 mL), brine (100mL) and dried over anhydrous Na₂SO₄. After evaporation of solvent invacuo the crude product was purified by flash silica columnchromatography using 0-10% Et₂O in hexane. The pure fractions werecombined and concentrated to obtain the mesylate 2 as colorless oil(30.6 g, 89%). ¹H NMR (400 MHz, CDCl₃): δ=5.42-5.21 (m, 4H), 4.20 (t,2H), 3.06 (s, 3H), 2.79 (t, 2H), 2.19-2.00 (m, 4H), 1.90-1.70 (m, 2H),1.06-1.18 (m, 18H), 0.88 (t, 3H). ¹³C NMR (CDCl₃): δ=130.76, 130.54,128.6, 128.4, 70.67, 37.9, 32.05, 30.12, 29.87, 29.85, 29.68, 29.65,29.53, 27.72, 27.71, 26.15, 25.94, 23.09, 14.60. MS. MW calc. forC₁₉H₃₆O₃S: 344.53; found: 343.52 [M−H].

Synthesis of (10Z,13Z)-10,13-Nonadecadienenitrile or Linoleyl-1-cyanide(3)

To a solution of the mesylate 2 (3.44 g, 10 mmol) in ethanol (90 mL), asolution of KCN (1.32 g, 20 mmol) in water (10 mL) was added and themixture was refluxed for 30 minutes. After cooling, completion ofreaction was confirmed by TLC. Water was added to the cooled reactionmixture and the product was extracted into ether (2×200 mL) followed bystandard work-up. The crude product thus obtained was purified by columnchromatography (0-10% Et₂O in hexanes) to obtain compound 3 as colorlessoil (2 g, 74%). ¹H NMR (400 MHz, CDCl₃): δ=5.33-5.22 (m, 4H), 2.70 (t,2H), 2.27-2.23 (m, 2H), 2.00-1.95 (m, 4H), 1.61-1.54 (m, 21-I),1.39-1.20 (m, 18H), 0.82 (t, 3H). ¹³C NMR (CDCl₃): δ=130.20, 129.96,128.08, 127.87, 119.78, 70.76, 66.02, 32.52, 29.82, 29.57, 29.33, 29.24,29.19, 29.12, 28.73, 28.65, 27.20, 27.16, 25.62, 25.37, 22.56, 17.10,14.06. MS. MW calc. for C₁₉H₃₃N, 275.47; found: 276.6 [M+H].

Synthesis of (6Z,9Z)-heptatriaconta-6,9-dien-19-one (6)

Freshly activated Mg turnings (0.144 g, 6 mmol) were charged into aflame dried 500 mL 2NRB flask equipped with a magnetic stir bar and areflux condenser. This set-up was degassed and flushed with argon and 10mL of anhydrous ether was added to the flask via syringe. Thecommercially available stearyl bromide 26 (2.65 g, 5 mmol) dissolved inanhydrous ether (10 mL) was added dropwise via syringe to the flask.After completing the addition, the reaction mixture was kept at 35° C.for 1 hour in a warm water bath and then cooled over an ice bath. Thecyanide 3 (1.38 g, 5 mmol) dissolved in anhydrous ether (20 mL) wasadded drop-wise into the reaction mixture with stirring. An exothermicreaction was observed and the mixture was allowed to stir overnight atambient temperature. The reaction was quenched by drop-wise additionacetone (10 mL), followed by ice cold water (60 mL). The reactionmixture was subsequently treated with aq. H₂SO₄ (10% by volume, 200 mL)until the solution became homogeneous and the layers were separated. Theproduct was extracted into ether (2×100 mL), followed by standardwork-up. The crude residue thus obtained was purified by columnchromatography using 0-0.7% ether in hexane as eluting system to obtaina pure ketone 6 as a colorless oil. ¹H-NMR (400 MHz, CDCl₃): δ=5.42-5.30(m, 4H), 2.79-2.78 (t, 2H), 2.40-2.37 (t, 4H), 2.08-2.03 (m, 4H),1.58-1.54 (m, 4H), 1.36-1.26 (br m, aliphatic protons), 0.91-0.87 (t,6H). IR (cm⁻¹): 2924, 2854, 1716, 1465, 1375, 721.

Synthesis of N-methylcyclopent-3-enamine 8

A solution of t-butyl 3-cyclopentenylcarbamate (7, 10 g, 0.0492 mol) inanhydrous THF (70 mL) was added slowly into a stirred suspension ofLiAlH₄ (3.74 g, 0.09852 mol) in THF (anhydrous, 200 mL) at 0° C. undernitrogen atmosphere. After completing the addition, the reaction mixturewas warmed to room temperature and then heated to reflux for 4 hours.Progress of the reaction was monitored by TLC. After completion ofreaction (by TLC), the mixture was cooled to 0° C. and quenched withcareful addition of saturated Na₂SO₄ solution. Reaction mixture wasstirred for 4 hours at room temperature and filtered off. Residue waswashed well with THF. The filtrate and washings were mixed and dilutedwith 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes atroom temperature. The volatilities were removed in vacuo to furnish thehydrochloride salt of 8 as a white solid. Yield: 7.12 g. ¹H-NMR (400MHz, DMSO-d₆): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60(m, 2H), 2.50-2.45 (m, 5H).

Synthesis of benzyl cyclopent-3-enyl(methyl)carbamate 9

NEt₃ (37.2 mL, 0.2669 mol) was added to a stirred solution of compound 8in 100 mL dry DCM in a 250 mL two neck RBF and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO₃solution (1×50 mL) followed by standard work-up. The crude was thensubjected to silica gel column chromatography to obtain 9 as a stickymass. Yield: 11 g (89%). ¹H-NMR (400 MHz, CDCl₃): δ=7.36-7.27 (m, 5H),5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H),2.30-2.25 (m, 2H). MS. MW calc. for C₁₄H₁₇NO₂: 231.13; found: 232.3[M+H].

Synthesis of benzyl (1S,3R,4S)-3,4-dihydroxycyclopentyl(methyl)carbamate(10)

The cyclopentene 9 (5 g, 0.02164 mol) was dissolved in a solution of 220mL acetone and water (10:1) in a single neck 500 mL RBF and to it wasadded N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2mL of 7.6% solution of OsO₄ (0.275 g, 0.00108 mol) in ten-butanol atroom temperature. After completion of the reaction (˜3 hours), themixture was quenched with addition of solid Na₂SO₃ and resulting mixturewas stirred for 1.5 hours at room temperature. Reaction mixture wasdiluted with DCM (300 mL) and washed with water (2×100 mL) followed bysaturated NaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally withbrine (1×50 mL). Organic phase was dried over an Na₂SO₄ and solvent wasremoved in vacuo. Silica gel column chromatographic purification of thecrude material afforded a mixture of diastereomers, which were separatedby preparative HPLC (Mobile phase A: 0.05% trifluoroacetic acid, mobilephase B: 100% acetonitrile, 0.01 min-22 min 20 mL/min, 80:20 A:B; 22min-46 min 16 mL/min, 80:20 A:B, 46 min 16 mL/min, 80:20 A:B). Theprocess yielded 6 grams of crude benzyl(1S,3R,4S)-3,4-dihydroxycyclopentyl(methyl)carbamate (10), shown below.

Diol 10-Peak-1:

White solid; 5.13 g (96%). ¹H-NMR (400 MHz, DMSO-d₆): δ=7.39-7.31 (m,5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m,2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS. MW calc. for C₁₄H₁₉NO₄:265.13; found: 266.3 [M+11], [M+NH₄+]-283.5 present, HPLC-97.86%.Stereochemistry was confirmed by X-ray.

Synthesis of Ketone 12:

A mixture of compound 10 (1.85 g, 7.8 mmol), ketone 6 (2.74 g, 5.2 mmol)and p-TSA (0.1 eq) was heated under toluene reflux with Dean-Starkapparatus for 3 hours. Removal of solvent in vacuo followed by columnchromatography afforded compound 12 (3.6 g, 93%) as a colorless oil.¹H-NMR (400 MHz, CDCl₃): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-ELSD: 98.65%.

Synthesis of amine 13

A solution of compound 12 (2 g, 2.68 mmol) in hexane (20 mL) was addedin a drop-wise fashion to an ice-cold solution of LAH in THE (1 M, 5.4mL). After completing the addition, the mixture was heated at 40° C.over 0.5 hour then cooled again on an ice bath. The mixture wascarefully hydrolyzed with saturated aqueous Na₂SO₄ then filtered throughcelite and solvent were removed in vacuo to obtain an oily residue.Silica gen column chromatography of the crude thus obtained gave pureamine 13 (1.32 g, 79%) as a colorless oil. ¹H-NMR (400 MHz, CDCl₃):δ=7.34-7.33 (m, 4H), 7.30-7.28 (m, 1H), 5.36-5.32 (m, 4H), 5.12 (s, 2H),4.77-4.70 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.75 (m, 5H), 2.04-2.00 (m,4H), 1.95-1.92 (m, 2H), 1.62 (m, 4H), 1.56-1.53 (m, 2H), 1.35-1.23 (brm, 40H), 0.87-0.84 (m, 6H). HPLC-ELSD: 98.34%.

Synthesis of(3aR,Ss,6aS)—N,N,N-trimethyl-2-((9Z,12Z)-octadeca-9,12-dienyl)-2-octadecyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-aminiumchloride, 14

Compound 13 (500 mg, 0.778 mmol) was dissolved inchloroform-acetonitrile (1:1) mixture (5 mL each) and bubbled withchloromethane (1.263 g, 16.14 mmol) for 1.5 minutes at ice bath. Thereaction mixture was then heated in a pressure bottle for 3 hours at100° C. The TLC showed complete disappearance of the starting lipid 13.Aqueous workup then silica gel column chromatography (0-20%CH₃OH/CH₂Cl₂) gave compound 14 (480 mg, 89%), shown below, as a whitewaxy solid.

¹H NMR (400 MHz, CDCl₃): δ 5.40-5.26 (m, 4H), 4.70 (d, J=3.2, 2H),3.80-3.61 (m, 1H), 3.48 (s, 9H), 2.75 (s, 2H), 2.35-2.20 (m, 2H),2.15-2.08 (m, 2H), 2.06-1.99 (m, 4H), 1.60 (d, J=8.4, 2H), 1.46 (d,J=7.2, 2H), 1.32-1.20 (m, 50H), 0.89-0.83 (m, 6H). ¹³C NMR (101 MHz,cdcl₃) δ 129.76, 129.74, 129.64, 129.60, 127.57, 127.52, 127.45, 112.77,73.59, 52.03, 35.50, 34.25, 34.20, 32.91, 31.46, 31.06, 29.36, 29.28,29.24, 29.20, 29.11, 29.06, 29.00, 28.90, 28.83, 26.76, 26.74, 25.18,23.97, 23.13, 22.23, 22.11, 13.66, 13.62. MS (LC-MS) MW calc. forC₄₅H₈₆NO₂ ⁺: 672.67; found 672.69.

Formulation of Transfection Reagents

Cationic lipid and colipids in chloroform were dried by a N₂ streamfollowed by vacuum-dessication to remove residual organic solvent. Thedried lipid film was hydrated using 10 mM HEPES buffer, pH 7.4 at 37° C.The formed liposomes were extruded to yield an average particle size of˜200 nm.

L8 DOPE CHOL Total Lipid excipients molar comp. (%) 48 47 5 100Excipient MW 672 744 386.7 Lipid weight (mg) 4.66 5.06 0.28 10.00 Lipid(wt %) 46.58 50.62 2.80 Volume taken from the stock 186 203 11 400solution (μL) Volume of buffer added to make 10.00 final formulation(mL)Excipient molar ratio: Cationic lipid (L8),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),Cholesterol-48:47:5

Each lipid component was dissolved in chloroform to a workingconcentration (25 mg/mL). Next, the desired amount of each excipientswas pipetted into a glass vial and thoroughly mixed. The chloroform wascarefully evaporated using an argon stream at ambient temperature insidea fume hood to make a thin film inside. The lipid residue was placed ona vacuum pump for 10-15 minutes to remove any residual organic solvent.

The vial was removed from the vacuum pump and the residue immediatelysuspended in calculated volume of 10 mM HEPES at pH 7.4 to obtain afinal lipid concentration of 1 mg/mL. The mixture was stirred in anagitator at 37° C. for 1 hr. The suspension was extruded (LIPEX™Extruder) using polycarbonate filters (two 0.2 μm filters and one 0.4 μmprefilter, Whatman Nuclepore Track-Etch Membrane). The final formulationwas filtered using a 0.45 μm sterile filter before use. Particle sizewas measured by dynamic light scattering. 10 μL of the final formulationwas diluted to 1 mL in pre-filtered PBS buffer (0.02 micron filter) formeasurement.

Testing of Transfection Reagents on Plated GFP-CHO Cells

Preparation of Compound 200

Compound 100 (500 mg, 0.778 mmol) was dissolved inchloroform:acetonitrile (1:1) mixture (5 mL each) and bubbled withchloromethane (1,263 g, 16.14 mmol) for 1.5 min while the reactionvessel was submerged in an ice bath. The reaction mixture was thensubjected to microwave synthesis for 2 h at 100° C. with 250 W powersupply and 247 psi pressure. Thin layer chromatography showed a completereaction. Aqueous workup and column chromatography (0-20% CH₃OH/CH₂Cl₂)gave compound 200, 480 mg, 89%, as a white waxy solid. ¹H NMR (400 MHz,CDCl₃) δ 5.41-5.28 (m, 8H), 4.12 (t, J=6.0, 2H), 3.95 (d, J=4.7, 1H),3.61-3.49 (m, 2H), 3.47 (s, 9H), 2.76 (t, J=6.4, 4H), 2.03 (q, J=6.7,8H), 1.96-1.83 (m, 2H), 1.55 (dd, J=14.0, 10.2, 4H), 1.37-1.22 (m, 36H),0.87 (t, J=6.8, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 130.50, 130.48, 130.42,130.37, 128.30, 128.25, 128.21, 128.19, 113.52, 72.82, 69.64, 64.83,53.75, 37.78, 37.12, 31.81, 30.18, 29.97, 29.94, 29.87, 29.82, 29.63,29.59, 28.20, 27.52, 27.49, 25.92, 24.33, 24.16, 22.86, 14.36. MS(LC-MS) m/z: Calcd for C₄₄H₈₂NO₂, the ammonium ion, 100%) 656.10. Found656.66.

Preparation of Compound 201

Compound 201 was prepared from compound 101, using the method same asfor preparation of compound 200. Aqueous workup then columnchromatography (0-20% CH₃OH/CH₂Cl₂) gave compound 201, 81%, as a whitewaxy solid. ¹H NMR (400 MHz, CDCl₃) δ 5.40-5.24 (m, 8H), 4.70 (d, J=3.3,2H), 3.77-3.67 (m, 1H), 3.47 (s, 9H), 2.75 (t, J=6.4, 4H), 2.25 (dd,J=13.1, 5.7, 2H), 2.12 (d, J=11.6, 2H), 2.03 (dd, J=13.6, 6.7, 8H), 1.60(d, J=8.2, 2H), 1.43 (dd, J=24.3, 6.3, 2H), 1.39-1.16 (m, 36H), 0.87 (t,J=6.7, 6H). ¹³C NMR (101 MHz, cdcl₃) δ 129.96, 129.94, 129.84, 129.80,127.78, 127.73, 127.65, 127.64, 112.96, 73.80, 52.26, 35.72, 34.43,33.10, 31.27, 29.57, 29.50, 29.43, 29.40, 29.32, 29.27, 29.21, 29.09,29.03, 26.97, 26.95, 25.38, 24.19, 23.35, 22.31, 13.82. MS (LC-MS) m/z:Calcd for C₄₅H₈₂NO₂, the ammonium ion, 100%) 668.10. Found 668.66.

Preparation of Compound 202

Compound 202 was prepared from compound 102, using the method same asfor preparation of compound 200. Aqueous workup then columnchromatography (0-20% CH₃OH/CH₂Cl₂) gave compound 202, 75.4%, as a whitewaxy solid. ¹H NMR (400 MHz, CDCl₃) δ 5.40-5.26 (m, 4H), 4.70 (d, J=3.2,2H), 3.80-3.61 (m, 1H), 3.48 (s, 9H), 2.75 (s, 2H), 2.35-2.20 (m, 2H),2.15-2.08 (m, 2H), 2.06-1.99 (m, 4H), 1.60 (d, J=8.4, 2H), 1.46 (d,J=7.2, 2H), 1.32-1.20 (m, 50H), 0.89-0.83 (m, 6H). ¹³C NMR (101 MHz,cdcl₃) δ 129.76, 129.74, 129.64, 129.60, 127.57, 127.52, 127.45, 112.77,73.59, 52.03, 35.50, 34.25, 34.20, 32.91, 31.46, 31.06, 29.36, 29.28,29.24, 29.20, 29.11, 29.06, 29.00, 28.90, 28.83, 26.76, 26.74, 25.18,23.97, 23.13, 22.23, 22.11, 13.66, 13.62. MS (LC-MS) m/z: Calcd forC₄₅H₈₆NO₂, the ammonium ion, 100%) 672.13. Found 672.69.

Preparation of Compound 203

Compound 203 was prepared from compound 103, using the method same asfor preparation of compound 200. Aqueous workup then columnchromatography (0-20% CH₃OH/CH₂Cl₂) gave compound 203, 94%, as a whitewaxy solid. ¹H NMR (400 MHz, CDCl₃) δ 5.44-5.23 (m, 8H), 4.89-4.77 (m,1H), 3.69-3.63 (m, 2H), 3.45 (s, 9H), 2.75 (t, J=6.4, 4H), 2.46 (t,J=6.6, 2H), 2.02 (dd, J=13.8, 6.9, 10H), 1.48 (d, J=5.8, 4H), 1.40-1.14(m, 36H), 0.87 (t, J=6.7, 6H). ¹³C NMR (101 MHz, cdcl₃) δ 171.55,129.86, 129.76, 127.64, 127.57, 75.31, 65.29, 53.03, 33.64, 31.18,29.58, 29.33, 29.20, 29.18, 29.00, 28.95, 26.89, 26.86, 25.29, 25.04,22.22, 18.08, 13.73. MS (LC-MS) m/z: Calcd for C₄₄H₈₂NO₂, the ammoniumion, 100%) 656.1. Found 656.65.

Preparation of Compound 204

Compound 204 was prepared from compound 104, using the method same asfor preparation of compound 200. Aqueous workup then columnchromatography (0-20% CH₃OH/CH₂Cl₂) gave compound 204, 78.3%, as a whitewaxy solid. ¹H NMR (400 MHz, CDCl₃) δ 5.78 (s, 1H), 5.40-5.27 (m, 8H),4.68-4.63 (m, 1H), 3.72 (s, 2H), 3.40 (s, 9H), 3.31-3.22 (m, 2H), 2.75(t, J=6.4, 4H), 2.03 (dd, J=13.7, 6.8, 10H), 1.49-1.38 (m, 4H),1.36-1.11 (m, 36H), 0.87 (t, J=6.7, 6H). ¹³C NMR (101 MHz, cdcl₃) δ157.28, 130.18, 130.10, 127.95, 127.89, 75.28, 64.75, 53.50, 37.95,34.31, 31.50, 29.67, 29.63, 29.57, 29.55, 29.32, 29.30, 27.22, 27.18,25.61, 25.37, 23.86, 22.55, 14.06. MS (LC-MS) m/z: Calcd for C₄₄H₈₃N₂O₂,the ammonium ion, 100%) 671.1. Found 671.65.

Preparation of Compound 205

Compound 205 was prepared from compound 105, using the method same asfor preparation of compound 200. Aqueous workup then columnchromatography (0-20% CH₃OH/CH₂Cl₂) gave compound 205, 91%, as a whitewaxy solid. ¹H NMR (400 MHz, CDCl₃) δ 5.44 (d, J=9.1, 1H), 5.40-5.26 (m,8H), 4.51 (s, 2H), 4.02-3.98 (m, 2H), 3.52 (s, 10H), 2.75 (t, J=6.4,4H), 2.02 (q, J=6.8, 8H), 1.42 (dd, J=16.2, 6.6, 4H), 1.38-1.20 (m,36H), 0.87 (t, J=6.8, 6H). ¹³C NMR (101 MHz, cdcl₃) δ 155.18, 130.50,130.40, 128.27, 128.20, 65.66, 58.15, 54.64, 52.22, 35.42, 31.81, 29.98,29.87, 29.64, 29.61, 27.53, 27.49, 26.29, 25.92, 22.86, 14.38. MS(LC-MS) m/z: Calcd for C₄₃H₈₁N₂O₂, the ammonium ion, 100%) 657.1. Found657.64.

Preparation of Compound 206

Compound 206 is prepared from compound 106, using the method same as forpreparation of compound 200. Aqueous workup then column chromatography(0-15% MeOH/DCM) gives compound 206.

Preparation of Compound 207

Compound 207 is prepared from compound 107, using the method same as forpreparation of compound 200. Aqueous workup then column chromatography(0-15% MeOH/DCM) gives compound 207.

Preparation of Compound 208

Compound 208 is prepared from compound 108, using the method same as forpreparation of compound 200. Aqueous workup then column chromatography(0-15% MeOH/DCM) gives compound 208.

Preparation of Compound 209

Compound 209 is prepared from compound 109, using the method same as forpreparation of Compound 200. Aqueous workup then column chromatography(0-15% MeOH/DCM) gives compound 209.

siRNA

Double-stranded siRNAs were synthesized by Alnylam Pharmaceuticals withantisense sequence 5′UCGAAGUACUCAGCGUAAGdTdT (target-luciferase) andsense sequence 5′CUUACGCUGAGUACUUCGAdTdT. Purification of thesynthesized oligoribonucleotides was achieved by anion exchange HPLC asper our established procedures. Double stranded siRNAs were obtained byannealing equimolar amounts of sense and antisense strands.

Preparation of Liposomes

Liposomes were prepared using a film hydration method whereby thecharged lipid and colipids in chloroform were dried into a thin film byN₂ flux followed by vacuum dessication for 10 min to remove residualorganic solvent. The dried lipid film was hydrated using 10 mM HEPESbuffer, pH 7.4 for 1 h in 37° C. shaker water bath. Formed multilamellarvesicles were vortexed with siRNA for 1 min and then extruded using aLIPEX™ extruder with polycarbonate membranes of sequential sizes (400nm, 200 nm, 200 nm) at 200-300 psi pressure, to form large unilamellarvesicles. The formed vesicles were sterile filtered using 0.45 μmfilter, prior to transfection and cell viability studies. The liposomeswere characterized using particle size analyser (Wyatt Technologies,Dyna ProTitan). All the formulations had uniform particle size of200-250 nm.

Cell Culture

HeLa (human cervical cancer cell line) cells stably transfected withluciferase enzymes were used. Cells were cultured in Dulbecco’ ModifiedEagle's medium (DMEM) with 10% heat inactivated fetal bovine serum (FBS)and antibiotics (Zeocin—0.5 mg/mL, Puromyin—0.5 μg/mL) at 37° C. with 5%CO₂ atmosphere.

Transfection Experiment

24 h before the experiment, HeLa cells (80-100% confluent) weretrypsinized and resuspended in fresh DMEM media with FBS (withoutanitibiotics). The cells were plated at density of 10,000 cells/well inwhite 96 well plates (View plate, 96 TC, Perkin Elmer). The plated cellswere incubated at 37° C. in 5% CO₂. On the day of experiment, liposomesand the siRNA were diluted in Opti-MEM I (Invitrogen) and mixed inappropriate proportions followed by incubation for 20 min with shakingto form liposome-siRNA complexes. Negative controls used were (i)untreated cells, and (ii) cells treated with only siRNA (lipid free).Positive controls were cells treated with siRNA complexes withLipofectamine 2000™ (LF2000), or DOTAP:DOPE (1:1). Other commerciallyavailable formulations namely Lipofectamine™, Lipofectin™, were alsotested.

In a second experiment HeLa cells are substituted with CHO cells ornon-adeherant suspension of CHO cells.

Gene-Knockdown Assay

Dual Luc Assay (Promega) was performed 22 h post-transfection todetermine the silencing efficiency of the liposomal formulations. Theprocedure was followed as per the manufacturer's protocol.

Liposomes were prepared using compounds 200-209 along with colipids DOPEand cholesterol. Formulation optimization was performed using compound200. Liposomes were prepared with 50-90 mol % compound 200, 10-50 mol %DOPE, and 0 or 10 mol % cholesterol. See Table 1. Dose response studieswere performed with these liposomes using 2 μg/mL lipid with siRNAconcentration varying in the range 10 μM to 1 μM.

TABLE 1 Code Compound 200 mol % DOPE mol % Chol mol % NF7 50 50 0 NF8 6040 0 NF9 70 30 0 NF10 80 20 0 NF11 90 10 0 NF12 50 40 10 NF13 60 30 10NF14 70 20 10 NF15 80 10 10 NF16 90 0 10

As seen from FIG. 1, formulations NF7 and NF12 had a higher silencingefficiency (60-75%, or expression of 40-25%) than other formulations.Also from FIG. 2 it was found the N/P ratio of 0.3 to 0.44 providedefficient transfection.

Formulations with the composition (mol %): charged lipid (45-63), DOPE(35-55), cholesterol (0-10) (H1-H11) were prepared (Table 2). As seenfrom FIG. 3, H1-H4, H8-H11 showed good silencing (70-75%) with N/Pbetween 0.3-0.39 as represented by % maximum knockdown (100−minimum %expression) (FIG. 4). On comparing all the efficient formulations ofcompound 200, it was found that all the formulations provided higher %efficiency than both Lipofectin and Lipofectamine™; and an efficiencybetween those of DOTAP:DOPE and LF2000™.

TABLE 2 Compound 200 DOPE Chol Code Mol % Mol % Mol % N/P H1 45.6 54.4 00.32 H2 48.1 51.9 0 0.33 H3 50.6 49.4 0 0.35 H4 53.1 46.9 0 0.37 H5 58.042.0 0 0.41 H6 60.5 39.5 0 0.42 H7 62.9 37.1 0 0.44 H8 52.7 37.3 10 0.39H9 52.9 42.1 5 0.38 H10 53.0 44.5 2.5 0.37 H11 47.9 47.1 5 0.34

Also to see the effect of particle size, H9 and H11 were formulated indifferent particle size (FIG. 5). A particle size in the range 200 nm to250 nm seemed to be optimal; larger size did not further improve thesilencing efficacy.

Formulations of other charged lipids (compounds 201-204) with similarcompositions were formulated (Table 3). These were designated asfollows: compound 201, K; compound 202, L; compound 203, M; compound204, P. All formulations were tested for transfection and cellviability, along with controls.

TABLE 3 Charged lipid DOPE Chol Total Series mol % mol % mol % mol % 145.56 54.44 0 100 2 48.08 51.92 0 100 3 50.60 49.40 0 100 4 53.10 46.900 100 5 52.73 37.27 10 100 6 52.92 42.08 5 100 7 53.01 44.49 2.5 100 847.94 47.06 5 100

From the results in FIG. 6A-CD, all the 32 formulations exceededLipofectin and Lipofectamine™ in terms of silencing efficacy. The bestcandidates for each of the charged lipid, were tested for transfectionefficiency using keeping LF2000 and DOTAP:DOPE as positive controls. Theresults (FIG. 7) obtained were the average from three independentexperiments in triplicate (n=9).

As shown in FIG. 8, formulations K2, and P5-P8 had a maximum knockdownhigher than LF2000 and much higher than DOTAP:DOPE. P7 was as potent asLF2000 and much more potent than DOTAP:DOPE (Table 4).

TABLE 4 IC₅₀ (nM) H1 NA K1 0.266 K2 0.38 L2 7.07 L8 0.932 M5 0.574 P50.108 P6 0.254 P7 0.054 P8 0.106 LF2000 0.0229 DOTAP 0.147

Dose response studies of compound 205 (designated R) formulations wereperformed with positive controls. The max knockdown of theR-formulations was higher than the controls (FIG. 9). More than 80%knockdown from all the R formulations (except R1) and almost 90%knockdown from formulations R5, R6 and R7 was achieved. These valueswere higher than LF2000 (75%) and DOTAP:DOPE (63%) at relatively lowsiRNA concentration of 10 nM (FIG. 10). Formulatins R1-R8 were moreefficient than LF2000 and DOTAP:DOPE. All the R-formulations were aspotent as LF2000 and 100-fold more potent than DOTAP:DOPE (Table 5).

TABLE 5 IC₅₀ (nM) R1 0.0998 R2 0.0275 R3 0.0136 R4 0.0265 R5 0.0122 R60.0104 R7 0.0203 R8 0.00915 LF2000 0.0148 DOTAP 0.918

Cell Viability Studies

Cell-Titer Blue assay (Promega) was used to determine cell viability ofthe lipoplexes. The assay is based on the ability of live cells toreduce non-fluorescent dye (resazurin, blue color) to its fluorescentmetabolite (resorufin, pink color). Cell viability was tested 22 h aftertransfection, by adding an appropriate volume of Cell Titer Blue™reagent to the cells and then incubating for 2.5 h at 37° C. Theread-out was obtained using a fluorometer with filter settings as 540 nm(excitation) and 590 nm (emission). Percentage cell viability wasobtained by normalizing to the untreated cells. Cell viabilityexperiments were performed three times in triplicate (n=9).

Most of the charged lipids used were toxic. Cell viability was alsotested over a range of lipid concentrations 0-60 μg/mL. From FIG. 11 andthe IC₅₀ values (lipid concentration at which 50% cells are viable)shown in Table 6, it was determined that the formulations (except L8 andM5) were safer than LF2000. In particular, compound 205 (R) formulationsR1, R2, R3 and R8 were notably safer than LF2000 (FIG. 12 and Table 7).

TABLE 6 IC₅₀ (μg/mL) H1 >100 K1 >100 K2 >60 L2 29.16 L8 19.18 M5 13.2 P534.16 P6 53.48 P7 >60 P8 >100 LF2000 16.82 DOTAP:DOPE >100

TABLE 7 IC₅₀ (μg/mL) R1 >100 R2 >100 R3 >60 R4 51.71 R5 17.47 R6 18.17R7 35.9 R8 >60 LF2000 16.82 DOTAP:DOPE >100

From the average of obtained results, compound 205 (R) formulations weremost efficient, while compound 202 (L) were least efficient. The rankorder was as follows (FIG. 13):

-   -   R>K˜P>H˜M>L

Nine different transfection agents (Table 1 and Scheme 12) andLipofectamine RNAiMax (Invitrogen) were used to deliver 1 nM of a potentsiRNA against GFP to a GFP-CHO cell line. RNAiMax was tested at 0.4μL/mL (concentration not available) and the nine transfection agentswere used at 0.5, 1, and 2.5 μg/mL. RNAiMax was used as a positivecontrol. Mixtures of transfection reagents and siRNA were made in blackoptical bottom 96 well plates and then cells were added. After 2 days,the relative GFP intensities were measured using a fluorescent platereader.

TABLE 1 Transfection reagents used in current study. FormulationCationic Lipid Number Mol % DOPE % Cholesterol % 1 48.08 51.92 — 2 47.9447.06 5 3 45.56 54.44 — 4 (K8) 47.94 47.06 5 5 (L8) 47.94 47.06 5 653.01 44.49 2.5 7 47.94 47.06 5 8 (P8) 47.94 47.06 5 9 47.94 47.06 5

Testing of Transfection Reagents on Suspended DG44 CHO Cells

The three active transfection agents, K8, L8 and P8, from the GFP-CHOtesting were used to transfect suspended CHO cells. 5 μL of 10 LDH-AsiRNA was added to a tube and 500 μL CD DG44 media added to it.Transfection reagent was added to the mixture, the tube mixed by pipetteaspiration and incubated at room temperature for 15 minutes. Then themixture was added to 49.5 mL of media containing 200,000 cells/mL. Theflask was incubated and shaken at 120 rpm for several days. LDH activitywas measured by VetTest 8008 slide analyzer.

40 L Transfection

DG44 cells were grown in Invitrogen CD DG44 media. To seed the 40 Lbioreactor, cells were taken from 4-1 L disposable bioreactors. Thestarting cell density in the 40 L of culture was 120,000 cells/mL. Thebioreactor was allowed to equilibrate, with the cells added for 1 hourprior to transfection. For transfection, 400 μL of LDH-A siRNA (100 uMstock solution) was added to 400 mL of media and mixed. Then 32 mL of 1mg/mL P8 reagent was added and again mixed. This was allowed to incubatefor 15 minutes at room temperature and then added to the 40 Lbioreactor. Cell density and viability were measured using a Vi-Cellcell counter. The efficiency of transfection was determined by measuringLDH activity using a VetTest 8008 slide analyzer.

Evaluation of Nine Transfection Agents for Uptake Efficiency in CHOCells in Shake Flasks

To gauge the effectiveness of the transfection agents at introducing anucleic acid into CHO cells, the transfection agents were used tointroduce a potent GFP siRNA into GFP-CHO cells. Compared to aneffective concentration of Lipofectamine RNAiMAx, three of thetransfection agents were active (FIG. 14). These formulations weredesignated K8, L8 and P8. No obvious cytotoxicity was observed with anyconcentration of any formulation.

As K8 was the most active formulation in the GFP-CHO cells, it wastested using DG44 CHO cells in 50 mL of culture in a 250 mL shake flaskand a potent LDH siRNA. A range of K8 concentrations was tested alongwith an effective concentration of Lipofectamine RNAiMAx. After 3 days,LDH activity was lower in cultures where K8 was used (FIG. 15). Therewas also a higher cell density in flasks that had 0.6 or 1.2 μg/mL of K8conpared to RNAiMAx. It appears that RNAiMAx inhibited growth of CHOcell in suspension compared to K8. The highest concentrations of K8reduced the cell density, even though the LDH activity was stillreduced.

Because some transfection reagents did not seem to have the sameactivity in shake flasks as in a 96 well plate, the 3 activetransfection reagent formulations were tested similarly in 50 mL of DG44culture in 250 mL shake flasks. Surprisingly, P8 which was onlymarginally active against GFP-CHO cells, performed the best usingsuspended DG44 cell culture (FIG. 16). After 5 days, 0.8 μg/mL of P8resulted in the most LDH activity knockdown. Also, it is significantthat the cell density in the presence of P8 was greater than or equal tocells without transfection reagent added. P8 at a final concentration of0.8 μg/mL has been used numerous times in smaller bioreactors and wouldbe tested in a 40 L system.

Evaluation of Cationic Lipid Formulation P8 for Uptake Efficiency in a40 L Bioreactor.

After seeding the 40 L bioreactor, the cells generally grew with adoubling time of approximately 24 hours and the cell viability was over98% (FIG. 17). The cells reached a peak concentration of 3.1×10⁶cells/mL at day 5 and then began to decline. As expected in this unfedbatch culture, by day 6 the cells were in decline.

The LDH activity declined as the cells were growing following seedingand transfection. The LDH activity was reduced ˜80%, even as the cellshad doubled over 3 times (FIG. 18). There was diminished LDH activitythrough the entire experiment. The diminished LDH activity suggests thatthe transfection was successful with no detectable toxicity in the CHOcells.

Evaluation of Cationic Lipid Formulation P8 for Uptake Efficiency in a 3L Bioreactor.

The same LDH-directed siRNA formulated with P8 (single dose, 1 nM finalconcentration) that was used in the 250 mL shake flask study wasevaluated in the 3 L bioreactor. After seeding the 3 L bioreactor anddosing with the P8-formulated LDH siRNA, the cells generally grew with adoubling time of approximately 30 h and the cell viability was over 97%(FIG. 19). The DG44 cells reached a peak concentration of 3.0×10⁶cells/mL at day 4.

The LDH activity declined during the cell growth phase following seedingand transfection. The LDH activity was reduced >80% even as the cellshad doubled 3 times (FIG. 20). The observed decrease in LDH activityfollowing a single siRNA dose suggested high uptake efficiency with nodetectable adverse effect on CHO cell growth or viability.

Cryoprotectants and Lyophilization General Protocol for Making theFormulation

To a glass vial, various amounts of a siRNA complexing lipid, a membranefusogenic lipid and cholesterol dissolved in chloroform were pipettedout. The lipid solutions were mixed thoroughly and evaporated thechloroform using an argon stream at ambient temperature inside a fumehood to make a thin film inside. The residual organic solvent wasremoved under vacuo. The filmy residue was suspended in a suitablebuffer containing a suitable cryprotectant to obtain a final desiredlipid concentration. The mixture was agitated at 37° C. for 1 hr. Thesuspension was then extruded one or more times (LIPEX™ Extruder) throughpolycarbonate filters with pore sizes corresponding to the desiredparticle size. The final formulation was filtered through a 0.45-μmfilter and stored under appropriate conditions until use.

General Protocol for the Storage of Formulation at 4° C.

To a glass vial, various amounts of a siRNA complexing lipid, a membranefusogenic lipid and cholesterol dissolved in chloroform were pipettedout. The lipid solutions were mixed thoroughly and evaporated thechloroform using an argon stream at ambient temperature inside a fumehood to make a thin film inside. The residual organic solvent wasremoved under vacuo. The filmy residue was suspended in a suitablebuffer containing a suitable cryprotectant to obtain a final desiredlipid concentration. The mixture was agitated at 37° C. for 1 hr. Thesuspension was then extruded one or more times (LIPEX™ Extruder) throughpolycarbonate filters with pore sizes corresponding to the desiredparticle size. The final formulation was filtered through a 0.45-μmfilter and stored at 4° C. before use.

General Protocol for the Storage of Formulation Under Frozen Conditions

To a glass vial, various amounts of a siRNA complexing lipid, a membranefusogenic lipid and cholesterol dissolved in chloroform were pipettedout. The lipid solutions were mixed thoroughly and evaporated thechloroform using an argon stream at ambient temperature inside a fumehood to make a thin film inside. The residual organic solvent wasremoved under vacuo. The filmy residue was suspended in a suitablebuffer containing a suitable cryprotectant to obtain a final desiredlipid concentration. The mixture was agitated at 37° C. for 1 hr. Thesuspension was then extruded one or more times (LIPEX™ Extruder) throughpolycarbonate filters with pore sizes corresponding to the desiredparticle size. The final formulation was filtered through a 0.45-μmfilter and stored frozen at −20 to −80° C. until use.

General Protocol for the Storage of Formulation as Freeze Dried Powder

To a glass vial, various amounts of a siRNA complexing lipid, a membranefusogenic lipid and cholesterol dissolved in chloroform were pipettedout. The lipid solutions were mixed thoroughly and evaporated thechloroform using an argon stream at ambient temperature inside a fumehood to make a thin film inside. The residual organic solvent wasremoved under vacuo. The filmy residue was suspended in a suitablebuffer containing a suitable cryprotectant to obtain a final desiredlipid concentration. The mixture was agitated at 37° C. for 1 hr. Thesuspension was then extruded one or more times (LIPEX™ Extruder) throughpolycarbonate filters with pore sizes corresponding to the desiredparticle size. The final formulation was filtered through a 0.45-μmfilter. The formulation was then lyophilized and the powder was storedat −20 to −80° C. until use. Particles were then resuspended in waterbefore complexation with nucleic acid. General protocol for thepreparation and storage of lipid transfection agent complexed with siRNAas freeze dried powder

To a glass vial, various amounts of a siRNA complexing lipid, a membranefusogenic lipid and cholesterol dissolved in chloroform were pipettedout. The lipid solutions were mixed thoroughly and evaporated thechloroform using an argon stream at ambient temperature inside a fumehood to make a thin film inside. The residual organic solvent wasremoved under vacuo. The filmy residue was suspended in a suitablebuffer containing a suitable cryprotectant to obtain a final desiredlipid concentration. The mixture was agitated at 37° C. for 1 hr. Thesuspension was then extruded one or more times (LIPEX™ Extruder) throughpolycarbonate filters with pore sizes corresponding to the desiredparticle size. The final formulation was filtered through a 0.45-μmfilter. Complexes of nucleic acid with the lipid particles were made byincubating the nucleic acid with the formulation. The formulation wasthen lyophilized and the powder was stored at −20 to −80° C. until use.Particles were then resuspended in water or cell culture media beforeusing for transfection.

TABLE 2 Lipid excipients required to prepare 10 mL P8 formulation with 1mg/mL total lipid Lipid P DOPE CHOL Total Lipid excipients molar comp.(%) 48 47 5 100 Excipient MW 672 744 386.7 Lipid weight (mg) 4.66 5.060.28 10.00 Lipid (wt %) 46.58 50.62 2.80 Volume (μL) taken from thestock 186 203 11 400 solution (25 mg/mL) Volume of buffer added to make10.00 final formulation (mL)

TABLE 3 Lipid excipients required to prepare 10 mL L8 formulation with 1mg/mL total lipid Lipid L DOPE CHOL Total Lipid excipients molar comp.(%) 48 47 5 100 Excipient MW 672 744 386.7 Lipid weight (mg) 4.66 5.060.28 10.00 Lipid (wt %) 46.58 50.62 2.80 Volume (μL) taken from thestock 186 203 11 400 solution (25 mg/mL) Volume of buffer added to make10.00 final formulation (mL)

Example 1 Preparation of P8 Formulation with Sucrose as Cryoprotectent

To a 10-mL glass vial, 120 μL Lipid P, 130 μL1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 6.44 mL 10 mM HEPES (pH 7.4) containing 10 wt % sucrose fora final lipid concentration of 1 mg/mL. The mixture was agitated at 37°C. for 1 hr. The suspension was then extruded through polycarbonatefilters (one 0.4-μm prefilter and two 0.2-μm filters, Whatman NucleporeTrack-Etch Membrane) in a LIPEX™ Extruder. The final formulation wasfiltered through a 0.45-μm filter and was stored at 4° C. until use.

Example 2 Preparation of L8 Formulation with Sucrose as Cryoprotectent

To a 10-mL glass vial, 120 μL Lipid L, 130 μL1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 6.44 mL 10 mM HEPES (pH 7.4) containing 10 wt % sucrose fora final lipid concentration of 1 mg/mL. The mixture was agitated at 37°C. for 1 hr. The suspension was then extruded through polycarbonatefilters (one 0.4-μm prefilter and two 0.2-μm filters, Whatman NucleporeTrack-Etch Membrane) in a LIPEX™ Extruder. The final formulation wasfiltered through a 0.45-μm filter and was stored at 4° C. until use.

Example 3 Preparation of P8 Formulation with Glucose as Cryoprotectent

To a 10-mL glass vial, 120 μL Lipid P, 130 μL1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 6.44 mL 10 mM HEPES (pH 7.4) containing 5 wt % glucose fora final lipid concentration of 1 mg/mL. The mixture was agitated at 37°C. for 1 hr. The suspension was then extruded through polycarbonatefilters (one 0.4-μm prefilter and two 0.2-μm filters, Whatman NucleporeTrack-Etch Membrane) in a LIPEX™ Extruder. The final formulation wasfiltered through a 0.45-μm filter and was stored at 4° C. until use.

Example 4 Storage of P8 formulation as a freeze-dried powder withsucrose

Forty μL of the formulation prepared by method described in Example 1was freeze dried and the white powder was stored at −80° C. until use.For the transfection experiment in a 50-mL scale, the powder wassuspended in 40 μl, of water which was then mixed with 5 μL of 10 μMsiRNA solution in PBS buffer diluted with 500 μl, CD DG44 media.

Example 5 Storage of P8 Formulation in Frozen Condition with Sucrose

Forty μL of the formulation prepared by method described in Example 1was stored frozen at −80° C. For the transfection experiment in a 50-mLscale, the mixture was thawed slowly at room temperature and then mixedwith 5 μL of 10 μM siRNA solution in PBS buffer diluted with 500 μL CDDG44 media.

Example 6 Storage of P8 Formulation as a Freeze Dried Powder withGlucose

Forty μL of the formulation containing 5 wt % glucose prepared by methoddescribed in Example 2 was freeze dried and the white powder was storedat −80° C. until use. For the transfection experiment in a 50-mL scale,the powder was suspended in 40 μL of water and was then mixed with 5 μLof 10 μM siRNA solution in PBS buffer diluted with 500 μL CD DG44 media.

Example 7 Storage of P8 Formulation Frozen with Glucose

Forty μL of the formulation containing 5 wt % glucose prepared by methoddescribed in Example 2 was stored frozen at −80° C. For the transfectionexperiment in a 50-mL scale, the mixture was thawed slowly at roomtemperature and then mixed with 5 μL of 10 μM siRNA solution in PBSbuffer diluted with 500 μL CD DG44 media.

Example 8 Preparation of P8 Formulation by Extrusion Through SyringeMembrane Filters

To a 10-mL glass vial, 120 μL Lipid P, 130 μL1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and chloroform was removed using anargon stream at ambient temperature inside a fume hood to make a thinfilm inside. The residual organic solvent was removed under vacuum. Thefilmy residue was suspended in 6.44 mL 10 mM HEPES (pH 7.4) containing10 wt % of sucrose for a final lipid concentration of 1 mg/mL. Themixture was agitated at 37° C. using a shaker for 1 hr. The dispersionwas then extruded through a PVDF membrane filter with 0.22-μm pore sizeat 60° C. The final formulation was filtered through a 0.45-μm filterand separated into three fractions. One fraction was stored at 4° C. Thesecond fraction was frozen at −80° C. The third fraction was frozen at−80° C. for 2 hours and then lyophilized in a freeze-drier.

Example 9 Preparation of Liposome/siRNA Complexes and Storage as aLyophilized Powder

Forty μL of the P8 formulation prepared by high-pressure extrusion or byPDVF syringe membrane filtration methods described in Examples 1 and 8,respectively, were mixed with 5 μl, of 10 μM siRNA solution in PBSbuffer. The mixture was incubated for 15 minutes at room temperature,frozen at −80° C. for 2 hours and then lyophilized in a freeze-drier.The resulting white powder was stored at −80° C. until use. For thetransfection experiment in a 50-mL scale, the powder was suspended in500 μL of CD DG44 media at room temperature.

Example 10 Preparation of L8/siRNA Lipoplex as a Freeze Dried Powder

Forty μL of the L8 formulation prepared by high-pressure extrusion or byPDVF syringe membrane filtration methods described in Examples 1 and 8,respectively, were mixed with 5 μL of 10 μM siRNA solution in PBSbuffer. The mixture was incubated for 15 minutes at room temperature,frozen at −80° C. for 2 hours and then lyophilized in a freeze-drier.The resulting white powder was stored at −80° C. until use. For thetransfection experiment in a 50-mL scale, the powder was suspended in500 μL of CD DG44 media at room temperature.

Example 11 Use of Compound 103 (See Scheme 12) (Rather than DOPE) as aFusogenic Lipid in the L8 Formulation

To a 10-mL glass vial, 60 μL Lipid L, 65 μL compound 103 and 3.5 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 3.2 mL 10 mM HEPES (pH 7.4) containing 10 wt % of sucrosefor a final lipid concentration of 1 mg/mL. The mixture was agitated at37° C. for 1 hr. The suspension was then extruded once throughpolycarbonate filters (one 0.4-μm prefilter and two 0.2-μm filters,Whatman Nuclepore Track-Etch Membrane) using a LIPEX™ Extruder. Thefinal formulation was filtered through a 0.45-μm filter and stored at 4°C. until use.

Example 12 Use of Compound 103 (See Scheme 12) as a siRNA ComplexationAgent at Acidic pH

In this example, a formulation of compound 103 with DOPE and cholesterolat acidic pH was prepared. The compound 103 lipid, which was cationic atthis pH, acted as an siRNA condensation agent, whereas DOPE wasfusogenic lipid. To a 10-mL glass vial, 60 μL MC-3, 65 μL1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 3.5 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 3.2 mL 25 mM sodium acetate buffer (pH 5.3) for a finallipid concentration of 1 mg/mL. The mixture was agitated at 37° C. for 1hr. The suspension was then extruded through polycarbonate filters (one0.4-μm prefilter and two 0.2-μm filters, Whatman Nuclepore Track-EtchMembrane) using a LIPEX™ Extruder. The final formulation was filteredthrough a 0.45-μm filter and stored at 4° C. until use.

Example 13 Use of Compound 110 as a siRNA Complexation Agent at AcidicpH

In this example a formulation of compound 110 with DOPE and cholesterolat acidic pH was prepared. The compound 110 lipid, which has a two-aminehead group, was cationic at acidic pH and acted as a siRNA condensationagent, whereas DOPE was fusogenic lipid. To a 10-mL glass vial, 60 μLcompound 110, 65 μL 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)and 3.5 μL cholesterol solution (25 mg/mL in chloroform) were added. Thelipid solutions were mixed thoroughly, and the chloroform was removedusing an argon stream at ambient temperature inside a fume hood. Theresidual organic solvent was removed under vacuum. The filmy residue wassuspended in 3.2 mL 25 mM sodium acetate buffer (pH 5.3) for a finallipid concentration of 1 mg/mL. The clear solution was agitated at 37°C. for 1 hr. The suspension was then extruded through polycarbonatefilters (one 0.4-μm prefilter and two 0.2-μm filters, Whatman NucleporeTrack-Etch Membrane) using a LIPEX™ Extruder. The final formulation wasfiltered through a 0.45-μm filter and stored at 4° C. until use.

Example 13 Use of C12-200 as a siRNA Complexation Agent at Acidic pH

In this example a formulation of C12-200 (see Love, K. T., et al.,“Lipid-like materials for low-dose, in vivo gene silencing,” PNAS 107,5, (2010), 1864-1869, which is incorporated by reference in itsentirety) with DOPE and cholesterol at acidic pH was prepared. TheC120-200 lipid, which has a five-amine head group, was cationic atacidic pH and acted as a siRNA condensation agent, whereas DOPE wasfusogenic lipid. To a 10-mL glass vial, 200 μL C12-200, 130 μL1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 8.44 mL of 25 mM sodium acetate buffer (pH 5.3) for a finallipid concentration of 1 mg/mL. The clear solution was agitated at 37°C. for 1 hr. The suspension was then extruded through polycarbonatefilters (one 0.4-μm prefilter and two 0.2-μm filters, Whatman NucleporeTrack-Etch Membrane) using a LIPEX™ Extruder. The final formulation wasfiltered through a 0.45-μm filter and stored at 4° C. until use.

Example 14 Preparation of L8 Formulation with DLPE as the FusogenicLipid

To a 10-mL glass vial, 120 μL Lipid L, 130 μL1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 6.44 mL 10 mM HEPES containing 10 wt % of sucrose at pH 7.4for a final lipid concentration of 1 mg/mL. The mixture was agitated at37° C. for 1 hr. The suspension was then extruded through polycarbonatefilters (one 0.4-μm prefilter and two 0.2-μm filters, Whatman NucleporeTrack-Etch Membrane) using a LIPEX™ Extruder. The final formulation wasfiltered through a 0.45-μm filter and stored at 4° C. until use.

Example 15 Storage of P8 Formulation as a Freeze Dried Powder withDextran as a Cryoprotectant

A 40 μL of the formulation containing 20% w/v dextran in 10 mM HEPESbuffer prepared by method described in Example 2 was freeze dried andthe white powder was stored at −80° C. until use. For the transfectionexperiment in a 50 mL scale, the powder was suspended in 40 μL of waterwhich was then mixed with 5 μL of 10 μM siRNA solution in PBS bufferdiluted with 500 μL CD DG44 media.

Example 16 Storage of P8 Formulation as a Freeze Dried Powder withPolyvinyl Pyrrollidone as a Cryoprotectant

A 40 μL of the formulation containing 20% w/v polyvinyl pyrrollidone in10 mM HEPES buffer prepared by method described in Example 2 was freezedried and the white powder was stored at −80° C. until use. For thetransfection experiment in a 50 mL scale, the powder was suspended in 40μL of water which was then mixed with 5 μL of 10 μM siRNA solution inPBS buffer diluted with 500 μL CD DG44 media.

Example 17 Storage of P8 Formulation as a Freeze Dried Powder with aMixture of Polyvinyl Pyrrollidone and Sucrose as a Cryoprotectant

A 40 μL of the formulation containing 20% w/v polyvinylpyrrollidone/sucrose mixture in 10 mM HEPES buffer prepared by methoddescribed in Example 2 was freeze dried and the white powder was storedat −80° C. until use. For the transfection experiment in a 50 mL scale,the powder was suspended in 40 μL of water which was then mixed with 5μL of 10 μM siRNA solution in PBS buffer diluted with 500 μL CD DG44media.

Example 18 Use of Novel Lipids Containing Protonatable Amine Groups atpH 7.4 as a siRNA Complexation Agent in a Formulation

In this example, a formulation of compound 111 with DOPE and cholesterolat physiological pH was prepared. The compound 111 lipid, which wascationic at this pH (pKa>7.4), acted as an siRNA condensation agent,whereas DOPE was fusogenic lipid. To a 10-mL glass vial, 120 μL compound111, 130 μL 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7μL cholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 6.4 mL 10 mM HEPES buffer (pH 7.4) containing 10 wt %sucrose for a final lipid concentration of 1 mg/mL. The mixture wasagitated at 37° C. for 1 hr. The suspension was then extruded throughpolycarbonate filters (one 0.4-μm prefilter and two 0.2-μm filters,Whatman Nuclepore Track-Etch Membrane) using a LIPEX™ Extruder. Thefinal formulation was filtered through a 0.45-μm filter and stored at 4°C. until use.

Example 19 Preparation of L8 Formulation with DLPC

To a 10-mL glass vial, 120 μL lipid L, 130 μL1,2-Dioleoyl-sn-glycero-3-phosphocholine (DLPC) and 7 μLcholesterolsolution (25 mg/mL in chloroform) were added. The lipid solutions weremixed thoroughly, and the chloroform was removed using an argon streamat ambient temperature inside a fume hood. The residual organic solventwas removed under vacuum. The filmy residue was suspended in 6.44 mL 10mM HEPES containing 10 wt % of sucrose at pH 7.4 for a final lipidconcentration of 1 mg/mL. The mixture was agitated at 37° C. for 1 hr.The suspension was then extruded through polycarbonate filters (one0.4-μm prefilter and two 0.2-μm filters, Whatman Nuclepore TrackEtchMembrane) using a LIPEX™ Extruder. The final formulation was filteredthrough a 0.45-μm filter and stored at 4° C. until use.

Example 20 Preparation of L8 Formulation with DLPC and DOPE as HelperLipids

To a 10-mL glass vial, 120 μL lipid L, 130 μL of a 1:1 mixture of1,2-Dioleoyl-sn-glycero-3-phosphocholine (DLPC) and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 6.44 mL 10 mM HEPES containing 10 wt % of sucrose at pH 7.4for a final lipid concentration of 1 mg/mL. The mixture was agitated at37° C. for 1 hr. The suspension was then extruded through polycarbonatefilters (one 0.4-μm prefilter and two 0.2-μm filters, Whatman NucleporeTrack-Etch Membrane) using a LIPEX™ Extruder. The final formulation wasfiltered through a 0.45-μm filter and stored at 4° C. until use.

Example 21 Preparation of L8 Formulation with DOPS as Helper Lipid

To a 10-mL glass vial, 120 μL lipid L, 130 μL of2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) and 7 μL cholesterolsolution (25 mg/mL in chloroform) were added. The lipid solutions weremixed thoroughly, and the chloroform was removed using an argon streamat ambient temperature inside a fume hood. The residual organic solventwas removed under vacuum. The filmy residue was suspended in 6.44 mL 10mM HEPES containing 10 wt % of sucrose at pH 7.4 for a final lipidconcentration of 1 mg/mL. The mixture was agitated at 37° C. for 1 hr.The suspension was then extruded through polycarbonate filters (one0.4-μm prefilter and two 0.2-μm filters, Whatman Nuclepore Track-EtchMembrane) using a LIPEX™ Extruder. The final formulation was filteredthrough a 0.45-μm filter and stored at 4° C. until use.

Example 22 Preparation of L8 Formulation with DLPC and DOPS as HelperLipids

To a 10-mL glass vial, 120 μL lipid L, 130 μL of a 1:1 mixture of1,2-Dioleoyl-sn-glycero-3-phosphocholine (DLPC) and2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) and 7 μL cholesterolsolution (25 mg/mL in chloroform) were added. The lipid solutions weremixed thoroughly, and the chloroform was removed using an argon streamat ambient temperature inside a fume hood. The residual organic solventwas removed under vacuum. The filmy residue was suspended in 6.44 mL 10mM HEPES containing 10 wt % of sucrose at pH 7.4 for a final lipidconcentration of 1 mg/mL. The mixture was agitated at 37° C. for 1 hr.The suspension was then extruded through polycarbonate filters (one0.4-μm prefilter and two 0.2-μm filters, Whatman Nuclepore Track-EtchMembrane) using a LIPEX™ Extruder. The final formulation was filteredthrough a 0.45-μm filter and stored at 4° C. until use.

Example 23 Preparation of L8 Formulation with DOPE and DOPS as HelperLipids

To a 10-mL glass vial, 120 μL lipid L, 130 μL of a 1:1 mixture of1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) and 7 μL cholesterolsolution (25 mg/mL in chloroform) were added. The lipid solutions weremixed thoroughly, and the chloroform was removed using an argon streamat ambient temperature inside a fume hood. The residual organic solventwas removed under vacuum. The filmy residue was suspended in 6.44 mL 10mM HEPES containing 10 wt % of sucrose at pH 7.4 for a final lipidconcentration of 1 mg/mL. The mixture was agitated at 37° C. for 1 hr.The suspension was then extruded through polycarbonate filters (one0.4-μm prefilter and two 0.2-μm filters, Whatman Nuclepore Track-EtchMembrane) using a LIPEX™ Extruder. The final formulation was filteredthrough a 0.45-μm filter and stored at 4° C. until use.

Example 24 Preparation of Formulation with Compound 102 (See Scheme 12)as siRNA Complexing Agent at Acidic pH

To a 10-mL glass vial, 120 μL compound 102, 130 μL1,2-Dioleoyl-sn-glycero-3-phosphocholine (DLPC) and 7 μL cholesterolsolution (25 mg/mL in chloroform) were added. The lipid solutions weremixed thoroughly, and the chloroform was removed using an argon streamat ambient temperature inside a fume hood. The residual organic solventwas removed under vacuum. The filmy residue was suspended in 6.44 mL 25mM sodium acetate buffer (pH 5.3) containing 10 wt % of sucrose at pH7.4 for a final lipid concentration of 1 mg/mL. The mixture was agitatedat 37° C. for 1 hr. The suspension was then extruded throughpolycarbonate filters (one 0.4-μm prefilter and two 0.2-μm filters,Whatman Nuclepore Track-Etch Membrane) using a LIPEX™ Extruder. Thefinal formulation was filtered through a 0.45-μm filter and stored at 4°C. until use.

Example 25 Preparation of Formulation with Compound 102 (See Scheme 12)as siRNA Complexing Agent at Acidic pH with DLPC and DOPE as HelperLipids

To a 10-mL glass vial, 120 μL compound 102, 130 μL of a 1:1 mixture of1,2-Dioleoyl-sn-glycero-3-phosphocholine (DLPC) and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 6.44 mL 25 mM sodium acetate buffer (pH 5.3) containing 10wt % of sucrose at pH 7.4 for a final lipid concentration of 1 mg/mL.The mixture was agitated at 37° C. for 1 hr. The suspension was thenextruded through polycarbonate filters (one 0.4-μm prefilter and two0.2-μm filters, Whatman Nuclepore Track-Etch Membrane) using a LIPEX™Extruder. The final formulation was filtered through a 0.45-μm filterand stored at 4° C. until use.

Example 26 Preparation of Formulation with Compound 102 (See Scheme 12)as siRNA Complexing Agent at Acidic pH with2-dioleoyl-sn-glycero-3-phosphatidylserine as Helper Lipid

To a 10-mL glass vial, 120 μL compound 102, 130 μL of2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) and 7 μL cholesterolsolution (25 mg/mL in chloroform) were added. The lipid solutions weremixed thoroughly, and the chloroform was removed using an argon streamat ambient temperature inside a fume hood. The residual organic solventwas removed under vacuum. The filmy residue was suspended in 6.44 mL 25mM sodium acetate buffer (pH 5.3) containing 10 wt % of sucrose at pH7.4 for a final lipid concentration of 1 mg/mL. The mixture was agitatedat 37° C. for 1 hr. The suspension was then extruded throughpolycarbonate filters (one 0.4-μm prefilter and two 0.2-μm filters,Whatman Nuclepore Track-Etch Membrane) using a LIPEX™ Extruder. Thefinal formulation was filtered through a 0.45-μm filter and stored at 4°C. until use.

Example 27 Preparation of Formulation with Compound 102 (See Scheme 12)as siRNA Complexing Agent at Acidic pH with DLPC and DOPS as HelperLipids

To a 10-mL glass vial, 120 μL compound 102, 130 μL of a 1:1 mixture of1,2-Dioleoyl-sn-glycero-3-phosphocholine (DLPC) and2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) and 7 μL cholesterolsolution (25 mg/mL in chloroform) were added. The lipid solutions weremixed thoroughly, and the chloroform was removed using an argon streamat ambient temperature inside a fume hood. The residual organic solventwas removed under vacuum. The filmy residue was suspended in 6.44 mL 25mM sodium acetate buffer (pH 5.3) containing 10 wt % of sucrose at pH7.4 for a final lipid concentration of 1 mg/mL. The mixture was agitatedat 37° C. for 1 hr. The suspension was then extruded throughpolycarbonate filters (one 0.4-μm prefilter and two 0.2-μm filters,Whatman Nuclepore Track-Etch Membrane) using a LIPEX™ Extruder. Thefinal formulation was filtered through a 0.45-μm filter and stored at 4°C. until use.

Example 28 Preparation of Formulation with Compound 102 (See Scheme 12)as siRNA Complexing Agent at Acidic pH with DOPE and DOPS as HelperLipids

To a 10-mL glass vial, 120 μL compound 102, 130 μL of a 1:1 mixture of1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) and 7 μL cholesterolsolution (25 mg/mL in chloroform) were added. The lipid solutions weremixed thoroughly, and the chloroform was removed using an argon streamat ambient temperature inside a fume hood. The residual organic solventwas removed under vacuum. The filmy residue was suspended in 6.44 mL 25mM sodium acetate buffer (pH 5.3) containing 10 wt % of sucrose at pH7.4 for a final lipid concentration of 1 mg/mL. The mixture was agitatedat 37° C. for 1 hr. The suspension was then extruded throughpolycarbonate filters (one 0.4-μm prefilter and two 0.2-μm filters,Whatman Nuclepore Track-Etch Membrane) using a LIPEX™ Extruder. Thefinal formulation was filtered through a 0.45-μm filter and stored at 4°C. until use.

Example 29 Preparation of Formulations Using a Mixture of Quaternizedand Nonquaternized Lipid

In this example, a new formulation of compound 102 and lipid L with DOPEand cholesterol at physiological pH was prepared. To a 10-mL glass vial,120 μL of 1:1 a mixture of compound 102 and lipid L, 130 μL1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7 μLcholesterol solution (25 mg/mL in chloroform) were added. The lipidsolutions were mixed thoroughly, and the chloroform was removed using anargon stream at ambient temperature inside a fume hood. The residualorganic solvent was removed under vacuum. The filmy residue wassuspended in 6.4 mL 10 mM HEPES buffer (pH 7.4) containing 10 wt %sucrose for a final lipid concentration of 1 mg/mL. The mixture wasagitated at 37° C. for 1 hr. The suspension was then extruded throughpolycarbonate filters (one 0.4-μm prefilter and two 0.2-μm filters,Whatman Nuclepore Track-Etch Membrane) using a LIPEX™ Extruder. Thefinal formulation was filtered through a 0.45-μm filter and stored at 4°C. until use.

Example 30 Storage of Formulations Prepared as Described in Examples19-29

The formulations (examples 19-29) containing sucrose as a cryoprotectiveagent were filtered through a 0.45-μm filter and separated into threefractions. One fraction was stored at 4° C. The second fraction wasfrozen at −80° C. The third fraction was frozen at −80° C. for 2 hoursand then lyophilized in a freeze-drier and stored at −80° C. until use.

Example 31 Storage of Formulations Prepared as Described in Examples19-29

The formulations (examples 19-29) containing a mixture of sucrose andpolyvinyl pyrrollidone as a cryoprotective agent were filtered through a0.45-μm filter and separated into three fractions. One fraction isstored at 4° C. The second fraction is frozen at −80° C. The thirdfraction is frozen at −80° C. for 2 hours and then lyophilized in afreeze-drier and stored at −80° C. until use.

Example 32 Preparation Formulations by Extrusion Through SyringeMembrane Filters

The aqueous suspensions containing mixture of lipids in compositions asdescribed in examples 19-29 were extruded through a PVDF membrane filterwith 0.22-μm pore size at 60° C. The final formulation were filteredthrough a 0.45-μm filter and separated into three fractions. Onefraction was stored at 4° C. The second fraction was frozen at −80° C.The third fraction was frozen at −80° C. for 2 hours and thenlyophilized in a freeze-drier and stored at −80° C. until use.

Example 33 Preparation of siRNA Lipoplexes as a Freeze Dried Powder

Forty μL of the formulations described in examples 19 to 29 were mixedwith 5 μL of 10 μM siRNA solution in PBS buffer. The mixture wasincubated for 15 minutes at room temperature, frozen at −80° C. for 2hours and then lyophilized in a freeze-drier. The resulting white powderwas stored at −80° C. until use. For the transfection experiment in a50-mL scale, the powder was suspended in 500 μl, of CD DG44 media atroom temperature.

TABLE 4 Transfection Agents-Formulation, storage, in vitro siRNAactivity and cell viability siRNA Formulation Activity Cryoprotectant+/− Storage Cell % LDH Excipient Name +/− Preparation siRNA conditionsReconstitution viability KD Lipid Sucrose − Extrusion − At 4° C. forSolution 25 P/DOPE/ (10 wt %) 3 days warmed to Chol in 37° C. 10 mM +Extrusion − At 4° C. for Solution 64 HEPES 3 days warmed to buffer 37°C. + Extrusion − Stored frozen Thawed and 72 at −80° C. warmed to for 3days 37° C. + Extrusion − Freeze Warmed to 63 dried and RT stored at−80° C. for 3 days − Filtered − At 4° C. for Solution 37 through 0.22 3days warmed to μm filter at 37° C. 60° C. + Filtered − At 4° C. forSolution 59 through 0.22 3 days warmed to μm filter at 37° C. 60° C. +Filtered − Stored frozen Thawed and 63 through 0.22 at −80° C. warmed toμm filter at for 3 days 37° C. 60° C. + Filtered − Freeze Warmed to 65through 0.22 dried and RT and mixed μm filter at stored at with siRNA60° C. −80° C. for in media 3 days + Filtered + Freeze Warmed to 95 74through 0.22 dried and RT and mixed μm filter at stored at with media60° C. −80° C. and tested after 4 hours + Filtered + Freeze Warmed to 8687 through 0.22 dried and RT and mixed μm filter at stored at with media60° C. −80° C. and tested after 3 weeks + Filtered + Freeze Warmed to 8671 through 0.22 dried and RT and mixed μm filter at stored at with media60° C. −80° C. and tested after 5 weeks Lipid + Filtered + Freeze Warmedto 95 89 L/DOPE/ through 0.22 dried and RT and mixed Chol in μm filterat stored at with media 10 mM 60° C. −80° C. and HEPES tested afterbuffer 4 hours + Filtered + Freeze Warmed to 77 66 through 0.22 driedand RT and mixed μm filter at stored at with media 60° C. −80° C. andtested after 3 weeks + Filtered + Freeze Warmed to 63 56 through 0.22dried and RT and mixed μm filter at stored at with media 60° C. −80° C.and tested after 5 weeks kD—knock down; RT—room temperature

TABLE 5 Transfection Agents-Formulation, storage, in vitro siRNAactivity and cell viability % LDH Particle reduction Formulation Storagesize (siRNA % cell excipients Buffer Preparation condition (nm) % PDactivity) viability compound 110/ 25 mM extrusion 4° C., 2 days 109 16.869.4 98 DOPE/Chol NaAcetate MC-3/DOPE/Chol 25 mM extrusion 4° C., 2 days198 16.5 70.3 99 NaAcetate Lipid L/compound 10 mM Hepes/ extrusion 4°C., 2 days 188 22 68.4 98 103/Chol 10 wt % sucrose Lipid L/ 10 mM Hepes/syringe freeze dried complex 268 15 89 97 DOPE/Chol(L8) + si 10 wt %sucrose filtration at −80° C., 2 days RNA complex at 60° C. Lipid P/ 10mM Hepes/ syringe freeze dried complex 216 17 40.7 97 DOPE/Chol(P8) + si10 wt % sucrose filtration at −80° C., 2 days RNA complex at 60° C.Lipid P/ 10 mM Hepes/ syringe 4° C., 3 days 150 23 64 95 DOPE/Chol(P8) 5wt % glucose filtration at 60° C. Lipid P/ 10 mM Hepes/ extrusion Freezedried powder 190 70 70 94 DOPE/Chol(P8) 5 wt % glucose at −80° C., 3days Lipid P/ 10 mM Hepes/ extrusion 4° C., 3 days 150 23 64 96DOPE/Chol(P8) 10 wt % sucrose Lipid P/ 10 mM Hepes/ extrusion Frozen at−80° C., 187 15 72 93 DOPE/Chol(P8) 5 wt % glucose 3 days Lipid P/ 10 mMHepes/ extrusion Freeze dried powder 297 86 63 95 DOPE/Chol(P8) 10 wt %sucrose at −80° C., 3 days Lipid P/ 10 mM Hepes/ syringe 4° C., 2 days268 15 88.3 97 DOPE/Chol(L8) 10 wt % sucrose filtration at 60° C. LipidP/ 10 mM Hepes/ syringe 4° C., 2 days 216 17 87.3 98 DOPE/Chol(P8) 10 wt% sucrose filtration at 60° C. Lipid L/ 10 mM Hepes/ extruded Freezedried powder 231 21 88.3 98 DOPE/Chol(L8) 10 wt % sucrose at −80° C., 2days Lipid L/ 10 mM Hepes/ syringe Freeze dried powder 268 15 87.3 98DOPE/Chol(L8) 10 wt % sucrose filtration at −80° C., 2 days at 60° C.Lipid P/ 10 mM Hepes/ syringe Freeze dried powder 216 17 84.7 97DOPE/Chol(P8) 10 wt % sucrose filtration at −80° C., 2 days at 60° C.Lipid P/ 10 mM Hepes/ extruded Freeze dried powder 220 15 78.3 97DOPE/Chol (P8) 10 wt % sucrose at −80° C., 2 days Lipid P/ 10 mM Hepes/syringe Frozen at −80° C., 268 15 73.7 98 DOPE/Chol(L8) 10 wt % sucrosefiltration 2 days at 60° C. C120-200/ 25 mM extrusion 4° C., 2 days 24815 80.4 94.8 DOPE/Chol NaAcetate (pH 5.3)

1. A compound having the formula:

wherein: R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkoxy,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl;

 represents a connection between L₂ and L₁ which is: (1) a single bondbetween one atom of L₂ and one atom of L₁, wherein L₁ is C(R_(x)) or N;L₂ is —CR₅R₆—, —N(Q)-, ═C(R₅)—, —C(O)N(Q)-, —C(O)O—, —N(Q)C(O)—,—OC(O)—, or —C(O)—; (2) a double bond between one atom of L₂ and oneatom of L₁; wherein L₁ is C; L₂ is —CR₅═, —N(Q)═, —N—, —O—N═, —N(Q)-N═,or —C(O)N(Q)-N═; (3) a single bond between a first atom of L₂ and afirst atom of L₁, and a single bond between a second atom of L₂ and thefirst atom of L₁, wherein L₁ is C; L₂ has the formula

 wherein X is the first atom of L₂, Y is the second atom of L₂,represents a single bond to the first atom of L₁, and X and Y are each,independently, selected from the group consisting of —O—, —S—, alkylene,—N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O, —OC(O)O—,—OS(O)(Q₂)O—, and —OP(O)(Q₂)O—; Z₁ and Z₄ are each, independently, —O—,—S—, —CH₂—, —CHR₅—, CHR₅, or —CR₅R₅—; Z₂ is CH or N; Z₃ is CH or N; orZ₂ and Z₃, taken together, are a single C atom; A₁ and A₂ are each,independently, —O—, —S—, —CH₂—, —CHR₅—, or —CR₅R₅—; each Z isindependently N, C(R₅), or C(R₃); k is 0, 1, or 2; each m,independently, is 0 to 5; each n, independently, is 0 to 5; where thevariables m and n in the ring containing the ring atoms Z, Z₂ and Z₃ aresuch that the ring has a total of 3, 4, 5, 6, 7 or 8 ring atoms; (4) asingle bond between a first atom of L₂ and a first atom of L₁, and asingle bond between the first atom of L₂ and a second atom of L₁,wherein (A) L₁ has the formula:

 wherein X is the first atom of L₁, Y is the second atom ofL₁, - - - - - represents a single bond to the first atom of L₂, and Xand Y are each, independently, selected from the group consisting of—O—, —S—, alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—,—C(O)O, —OC(O)O—, —OS(O)(Q₂)O—, and —OP(O)(Q₂)O—; T₁ is CH or N; T₂ isCH or N; or T₁ and T₂ taken together are C═C; L₂ is CR₅; or (B) L₁ hasthe formula:

 wherein X is the first atom of L₁, Y is the second atom ofL₁, - - - - - represents a single bond to the first atom of L₂, and Xand Y are each, independently, selected from the group consisting of—O—, —S—, alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—,—C(O)O, —OC(O)O—, —OS(O)(Q₂)O—, and —OP(O)(Q₂)O—; T₁ is —CR₅R₅—, —N(Q)-,—O—, or —S—; T₂ is —CR₅R₅—, —O—, or —S—; L₂ is CR₅ or N; R₃ has theformula:

 wherein each of Y₁, Y₂, Y₃, and Y₄, independently, is alkyl,cycloalkyl, aryl, aralkyl, or alkynyl; or any two of Y₁, Y₂, and Y₃ aretaken together with the N atom to which they are attached to form a 3-to 8- member heterocycle; or Y₁, Y₂, and Y₃ are all be taken togetherwith the N atom to which they are attached to form a bicyclic 5- to 12-member heterocycle; each R_(n), independently, is H, halo, cyano,hydroxy, amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, orheterocyclyl; L₃ is a bond, —N(Q)-, —O—, —S—, —(CR₅R₆)_(a)—, —C(O)—, ora combination of any two of these; L₄ is a bond, —N(Q)-, —O—, —S—,—(CR₅R₆)_(a)—, —C(O)—, or a combination of any two of these; L₅ is abond, —N(Q)-, —O—, —S—, —(CR₅R₆)_(a)—, —C(O)—, or a combination of anytwo of these; each occurrence of R₅ and R₆ is, independently, H, halo,cyano, hydroxy, amino, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, orheterocyclyl; or two R₅ groups on adjacent carbon atoms are takentogether to form a double bond between their respective carbon atoms; ortwo R₅ groups on adjacent carbon atoms and two R₆ groups on the sameadjacent carbon atoms are taken together to form a triple bond betweentheir respective carbon atoms; each a, independently, is 0, 1, 2, or 3;wherein an R₅ or R₆ substituent from any of L₃, L₄, or L₅ is optionallytaken with an R₅ or R₆ substituent from any of L₃, L₄, or L₅ form a 3-to 8-member cycloalkyl, heterocyclyl, aryl, or heteroaryl group; and anyone of Y₁, Y₂, or Y₃, is optionally taken together with an R₅ or R₆group from any of L₃, L₄, and L₅, and atoms to which they are attached,form a 3- to 8-member heterocyclyl group; each Q, independently, is H,alkyl, acyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl orheterocyclyl; and each Q₂, independently, is O, S, N(Q)(Q), alkyl oralkoxy.
 2. The compound of claim 1 represented by formula I:

wherein, X and Y are each independently —O—, —S—, alkylene, —N(Q)-,—C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O, —OC(O)O—,—OS(O)(Q₂)O—, or —OP(O)(Q₂)O—; Q is H, alkyl, ω-aminoalkyl,ω-(substituted)aminoalkyl, ω-phosphoalkyl, or ω-thiophosphoalkyl; Q₂ isindependently for each occurrence O, S, N(Q)(Q), alkyl or alkoxy; A₁ andA₂ are each independently —O—, —S—, —CH₂—, —CHR⁵—, —CR⁵R⁵—; Z is N orC(R₃); each R′, R″, and R′″, independently, is H, alkyl, alkyl,heteroalkyl, aralkyl, cyclic alkyl, or heterocyclyl; R⁵ is H, halo,cyano, hydroxy, amino, optionally substituted alkyl, optionallysubstituted alkoxy, or optionally substituted cycloalkyl; and R₁, R₂,R₃, m and n are as defined in claim
 1. 3. The compound of claim 2,selected from the group consisting of:


4. The compound of claim 1 represented by formula XXXIII:

wherein, E is each independently for each occurrence —CH₂—, —O—, —S—,—SS—, —CO—, —C(O)O—, O(CO)—, —C(O)N(R′)—, —OC(O)N(R′)—,—N(R′)C(O)N(R″)—, —C(O)—N(R′)—N═C(R′″)—; —N(R′)—N═C(R″)—, —O—N═C(R″)—,—C(S)O—, —C(S)N(R′)—, —OC(S)N(R)—, —N(R′)C(S)N(R″)—,—C(S)—N(R′)—N═C(R′″); —S—N═C(R″); —C(O)S—, —SC(O)N(R′)—, —OC(O)—,—N(R′)C(O)—, —N(R′)C(O)O—, —C(R′″)═N—N(R′)—; —C(R′″)═N—N(R′)—C(O)—,—C(R′″)═N—O—, —OC(S)—, —SC(O)—, —N(R′)C(S)—, —N(R′)C(S)O—, —N(R′)C(O)S—,—C(R′″)═N—N(R′)—C(S)—, —C(R′″)═N—S—, C[═N(R′)]O, C[═N(R′)]N(R″),—OC[═N(R′)]—, —N(R″)C[═N(R′)]N(R′″)—, —N(R″)C[═N(R′)]—,

 arylene, heteroarylene, cycloalkylene, or heterocyclylene; each R′, R″,and R′″, independently, is H, alkyl, alkyl, heteroalkyl, aralkyl, cyclicalkyl, or heterocyclyl; and R₁, R₂ and R₃ are as defined in claim
 1. 5.The compound of claim 4, wherein E is O(CO), (CO)O, OC(O)N(R′), orN(R′)C(O)O.
 6. The compound of claim 2, selected from the groupconsisting of:


7. A method for delivering a nucleic acid to a cell comprisingcontacting cells with a compound according to claim
 1. 8. The method ofclaim 7, wherein the composition further comprises a lipid capable ofreducing aggregation.
 9. The method of claim 7, wherein the compositionfurther comprises a nucleic acid.
 10. The method of claim 7, wherein thecell is in suspension.
 11. The method of claim 10, wherein the volume ofthe suspension is at least 0.050 L.
 12. The method of claim 10, whereinthe volume of the suspension is at least 3 L.
 13. The method of claim10, wherein the volume of the suspension is at least 25 L.
 14. Themethod of claim 10, wherein the volume of the suspension is at least 40L.
 15. The method of claim 9, wherein the nucleic acid includes achemically modified nucleic acid.
 16. The method of claim 9, wherein thenucleic acid is 10 to 50 nucleotides long.
 17. The method of claim 9,wherein the nucleic acid is an oligonucleotide.
 18. The method of claim17, wherein the oligonucleotide is 10 to 50 nucleotides long.
 19. Themethod of claim 18, wherein the oligonucleotide is double stranded. 20.The method of claim 18, wherein the oligonucleotide is single stranded.21. The method of claim 9, wherein the nucleic acid is siRNA.
 22. Themethod of claim 9, wherein the nucleic acid is mRNA.
 23. The method ofclaim 9, wherein the nucleic acid is an antisense nucleic acid, amicroRNA, an antimicro RNA, an antagomir, a microRNA inhibitor, or animmune stimulatory nucleic acid.
 24. A storage-stable compositioncomprising a cryoprotectant selected from sucrose, trehalose, glucose,2-hydroxypropyl-α-cyclodextrin, and sorbitol, and a compound accordingto claim
 1. 25. The composition of claim 24, further comprising aneutral lipid.
 26. The composition of claim 24, further comprising asterol.
 27. The composition of claim 24, further comprising a lipidcapable of reducing aggregation.
 28. The composition of claim 24,further comprising a nucleic acid.
 29. The composition of claim 24,wherein the cryoprotectant is present at from 5 wt % to 25 wt %.
 30. Thecomposition of claim 24, wherein the cryoprotectant is present at from 7wt % to 15 wt %.
 31. The composition of claim 24, wherein thecryoprotectant includes sucrose.
 32. The composition of claim 24,wherein the composition is lyophilized.
 33. The composition of claim 28,wherein the composition is lyophilized.
 34. A method for reconstitutingthe composition of claim 32, comprising resuspending the composition ina liquid.
 35. The method of claim 34, further comprising adding a lipidto the resuspended composition.
 36. The method of claim 34, furthercomprising adding a nucleic acid to the resuspended composition.